WO2024083985A1 - Novel peptides and use thereof for modulating protein accumulation - Google Patents

Novel peptides and use thereof for modulating protein accumulation Download PDF

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WO2024083985A1
WO2024083985A1 PCT/EP2023/079154 EP2023079154W WO2024083985A1 WO 2024083985 A1 WO2024083985 A1 WO 2024083985A1 EP 2023079154 W EP2023079154 W EP 2023079154W WO 2024083985 A1 WO2024083985 A1 WO 2024083985A1
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protein
seq
cpep
plant cell
relates
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PCT/EP2023/079154
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French (fr)
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Jean-Philippe COMBIER
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Université Toulouse III - Paul Sabatier
Centre National De La Recherche Scientifique
Micropep Technologies
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Publication of WO2024083985A1 publication Critical patent/WO2024083985A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology

Definitions

  • the present invention relates to new peptides (cPEPs & altPEPs), their preparation method and their use to modulate the accumulation of specific proteins.
  • peptides are short sequences of 2 to approximately 100 amino acids. These are often very active molecules, such as hormones or venom compounds.
  • peptides In plants, peptides perform many biological functions, such as development or defense mechanisms. Because peptide detection is relatively difficult, only a limited number of peptides have been identified, likely underestimating the quantity and role of peptides in these organisms.
  • miPEPs small open reading frames encoding regulatory peptides. Nature. 2015 Apr 2;520(7545):90-3.
  • the miPEPs are produced in the same place as the miRs from which they come and help improve the transcription of the corresponding pri-miRs.
  • the activity of a miPEP is very specific to the corresponding mi R, making it possible to upregulate the chosen mi R without affecting the expression of other miRs.
  • the use of miPEPs can make it possible to modulate the expression of a gene, if it is regulated by a miR, itself regulated by a miPEP (WO 2015/063431).
  • the presentation below offers a universal, and easily exploitable, means for specifically modulating the accumulation of a chosen protein in a plant using either a non-natural peptide (invention No. 1; cPEP), that is to say a peptide which is not naturally produced by the plant, or a natural peptide (invention No. 2; altPEP), that is to say a peptide which is naturally produced by the plant.
  • cPEP non-natural peptide
  • altPEP that is to say a peptide which is naturally produced by the plant.
  • one of their aspects is to propose a process for the preparation and determination of a “cPEP” or “altPEP” peptide capable of modulating the accumulation (expression) of a protein. specific in a plant cell.
  • a second aspect of these is to propose a method of modulating the accumulation of a protein in a plant using a cPEP or an altPEP.
  • a third aspect of these is to propose the use of a cPEP or an altPEP to modulate the accumulation of a protein in a plant.
  • a fourth aspect of these is to propose a process to promote, slow down or prevent the development of a plant.
  • a fifth aspect of these is to propose cPEP peptides or altPEP peptides making it possible to modulate the accumulation of a protein in a plant.
  • Other complementary aspects of these inventions relate to a nucleic acid encoding a cPEP or an altPEP, compositions comprising a cPEP or an altPEP and modified or transgenic plants comprising a cPEP or an altPEP.
  • a first aspect thereof relates to a method for preparing and determining a cPEP, said cPEP: having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids ; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a. a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein; b. a step of determining within this mRNA the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein; vs.
  • mRNA messenger RNA
  • a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is a cPEP capable of modulating the accumulation of said protein in a plant cell.
  • the present invention is based on the unexpected observation made by the Inventors that it is possible to specifically modulate the accumulation of a protein using a particular peptide not produced naturally, the sequence of which corresponds to (artificial) translation of a fragment of messenger RNA (mRNA) encoding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
  • mRNA messenger RNA
  • cPEP complementary peptide designates an artificial peptide capable of specifically modulating the accumulation of a protein once introduced into a plant cell.
  • a cPEP is not naturally present in a plant cell. This means that the plant cell contains the cPEP information but does not contain the nucleic sequence capable of allowing its expression. Only the peptide sequence of cPEP can be deduced from the sequence of the mRNA coding said protein whose accumulation we want to modulate. A cPEP is only present in a plant cell once it has been introduced there in the form of a peptide or in the form of a nucleic acid encoding said peptide.
  • cPEP The specificity of cPEP towards a target protein (target gene) is determined by its amino acid sequence. Indeed, the sequence of a cPEP corresponds to the in silico (artificial) translation of a fragment of the mRNA coding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
  • the mRNA fragment used to determine the cPEP sequence can be chosen from the three existing reading frames on the mRNA sequence.
  • a fragment can be selected in reading frames +1, +2 or +3.
  • the three reading frames contain the information of a cPEP as it is possible that only one of the three reading frames (the +1, the +2 or the +3) or two of the three reading frames (+1 and +2, +1 and +3, or +2 and +3) contain the information of a cPEP.
  • reading frame designates the grouping of nucleotides constituting a nucleic acid sequence into consecutive triplets (or codons), which follow one another without interruption or overlap.
  • cPEPs have a size of 4 to 70 amino acids, in particular a size of 4 to 41 amino acids, in particular a size of 5 to 41 amino acids.
  • sequence of a cPEP corresponds to the translation of a fragment comprised of 4 to 70 nucleotide triplets, in particular a fragment comprised of 4 to
  • nucleotide triplets in particular a fragment comprising 5 to 40 nucleotide triplets, 7 to 20 nucleotide triplets or more particularly a fragment comprising 8 to 15 nucleotide triplets.
  • sequence of a cPEP corresponds to the translation into amino acids of a fragment of “3n” nucleotides of the mRNA of the target protein, n being between 4 and 70, in particular included from 4 to 41, in particular included from 5 to 40, from 7 to 20 or more particularly included from 8 to 15.
  • the cPEPs have a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids, and correspond respectively to the translation of fragments 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84 , 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 138, 141, 144, 147, 150, 153, 156,
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said fragment has a size of 3n nucleotides, n being included: from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said peptide has a size chosen from: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said cPEP has a size smaller than that of said protein (/e. that of which the cPEP modulates the 'accumulation).
  • cPEPs have the capacity to specifically modulate the accumulation of a protein without the accumulation of the mRNA corresponding to it being impacted.
  • the addition of a cPEP in a plant cell does not modify the quantity of mRNA making it possible to express the protein that it has the capacity to regulate, but only the quantity of said protein.
  • the term “protein” designates a sequence of amino acids whose information is encoded by a gene present on the genome of a plant cell.
  • gene we therefore designate, in particular, the nucleic acid sequence necessary for the synthesis of said protein.
  • a gene includes more than the nucleotides encoding the amino acid sequence of the protein.
  • a gene includes the DNA sequences necessary for the synthesis of a pre-messenger (pre-mRNA), which is then processed by the cellular machinery into messenger RNA (mRNA). The latter can then be translated, via ribosomes, into a protein.
  • pre-mRNA pre-messenger
  • mRNA messenger RNA
  • pre-messenger RNA has not undergone splicing and is likely to contain introns
  • mature messenger RNA may have undergone a splice and contains only exons.
  • a cPEP capable of modulating the accumulation of a protein
  • the “modulation” of the accumulation of a protein designates either an increase in the accumulation of said protein (/e. an increase in the quantity of protein in the plant cell), or a decrease in the accumulation of said protein (/e. a decrease in the quantity of protein in the plant cell).
  • one embodiment of the invention relates to the method of preparing and determining a cPEP as described above, in which said modulation of the accumulation of said protein induced by said cPEP is: a reduction of the accumulation of said protein; or an increase in the accumulation of said protein.
  • the increase and decrease in the accumulation of said protein can be measured and monitored using methods well known to those skilled in the art, such as the coupling of the protein to a marker via the use of cassettes. particular expression, or a Western blot.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which in step e., the quantity of protein in the presence of said peptide is greater than the quantity of protein in the absence of said peptide.
  • the expression of the corresponding mRNA is increased, which leads to greater production of the protein without modifying the quantity of said mRNA.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which in step e., the quantity of protein in the presence of said peptide is less than the quantity of protein in the absence of said peptide.
  • the quantity of protein in the presence of said peptide is less than the quantity of protein in the absence of said peptide.
  • the fragment of mRNA encoding said cPEP is located within the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein, said fragment as such is not translated naturally in said plant cell. This, regardless of the reading frame used.
  • the existence of a cPEP within said plant cell is therefore artificial and originates from human action. For this, it is possible either to artificially introduce said cPEP as such, or to introduce an expression cassette comprising the nucleic acid sequence encoding said cPEP and the means of expressing it in said plant cell.
  • the nucleic acid sequence naturally translated in said plant cell which comprises a fragment carrying the information of a cPEP
  • This sequence therefore corresponds to the main open reading frame, which codes the protein whose accumulation we want to modulate.
  • open reading frame and “ORF” (open reading frame) are equivalent, and can be used interchangeably. They correspond to a sequence of nucleotides (nucleic acids) in a DNA or RNA molecule that can potentially encode a peptide or a protein: said open reading frame begins with a START codon (the START codon generally encoding a methionine), followed by a series of codons (each codon encoding an amino acid), and ends with a STOP codon (the STOP codon not being translated).
  • START codon the START codon generally encoding a methionine
  • a series of codons each codon encoding an amino acid
  • STOP codon the STOP codon not being translated
  • the coding region of the mRNA therefore corresponds to the genetic sequence delimited by the START codon or initiation codon (most often encoding a methionine) at the 5' end and by the STOP codon at the 3' end.
  • the coding region of the mRNA therefore does not include the intronic sequences possibly present in the sequence of a gene or pre-messenger RNA (pre-mRNA), nor the 5'UTR and 3'UTR regions, because those -these are not translated and therefore do not encode part of the functional protein of the gene.
  • the sequence of a cPEP is determined by carrying out an (artificial) translation of a fragment of the mRNA of the protein whose accumulation we want to modulate, said fragment being chosen from the (coding) sequence of nucleic acids naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
  • the same nucleic acid sequence naturally translated in said plant cell can give different cPEPs depending on the mRNA fragment chosen.
  • this same mRNA fragment can also give different cPEPs depending on the reading frame used to translate it (artificially), i.e. depending on the grouping of the nucleotides of the sequence into consecutive triplets. Indeed and as previously mentioned, a translation can be carried out in the three different reading frames thus potentially leading to three different cPEPs.
  • the “+1” reading frame corresponds to the reading frame determined by the initiation codon of the protein, i.e. the START codon of the open reading frame used naturally for the translation of the mRNA.
  • the cPEP obtained has a sequence identical to that of a fragment of the acid sequence amino acids of the protein naturally encoded by said mRNA.
  • the “+2” and “+3” reading frames correspond to reading frames which are not (or little) used naturally for mRNA translation.
  • the “+2” reading frame corresponds to the reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to the open reading frame used naturally for the translation of the mRNA and the protein it encodes.
  • the “+3” reading frame corresponds to the reading frame shifted by two nucleotides at 3' (or one nucleotide at 5') relative to the open reading frame used naturally for the translation of the mRNA and the protein it encodes.
  • the cPEPs obtained have a sequence different from that of a fragment of the amino acid sequence. of the protein naturally encoded by said mRNA.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and in which said fragment is chosen: either in the same reading frame as the open reading frame encoding said protein; either in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and in which said fragment is chosen: either in the same reading frame as the open reading frame encoding said protein; either in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein.
  • a cPEP comprises either an AUG codon (and no STOP codon), or a STOP codon (and no AUG codon), or neither of these two elements.
  • the missing element or these missing elements it is the person skilled in the art who adds, if necessary, the missing element or these missing elements to allow, in a non-limiting manner, either to produce a cPEP in vitro by means of, for example, a microorganism then use it (in a composition for example), or introduce the sequence and the means of expressing it via a vector in a plant cell or a plant.
  • the invention therefore relates to the method for preparing and determining a cPEP as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention also relates to the method for preparing and determining a cPEP as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention relates to the method of preparing and determining a cPEP as described above, said cPEP: having a size ranging from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a. a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein; b. a step of determining within this mRNA the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein; vs. a step of determining within this nucleic acid sequence naturally translated in said plant cell a non-naturally translated fragment thereof, said non-naturally translated fragment:
  • mRNA messenger RNA
  • n being between 4 and 70, in particular n being between 4 and 41,
  • a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is a cPEP capable of modulating the accumulation of said protein in a plant cell.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame. of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open frame. reading of said protein.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or of two nucleotides 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in 3' (or of a nucleotide at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5 ') or two nucleotides 3' (or one nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said cPEP is a hydrophobic peptide or a hydrophilic peptide.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said cPEP is a hydrophobic peptide.
  • hydrophobic peptide is meant a peptide whose amino acid sequence comprises more than 50% hydrophobic amino acids.
  • more than 50% we mean that the amino acid sequence comprises more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80% of amino acids. hydrophobic.
  • more than 50% we also mean that the amino acid sequence comprises at least 51%, at least 56%, at least 61%, at least 66%, at least 71%, at least 76% or at least 81% hydrophobic amino acids.
  • hydrophobic amino acids is meant the amino acids chosen from: alanine (Ala / A), isoleucine (Ile / 1), leucine (Leu / L), methionine (Met / M), phenylalanine (Phe / F), tryptophan (Trp/W), tyrosine (Tyr/Y) and valine (Val/V).
  • the invention also relates to the method for preparing and determining a cPEP as described above, in which said cPEP is a hydrophilic peptide.
  • hydrophilic peptide is meant a peptide whose amino acid sequence comprises more than 50% hydrophilic amino acids.
  • more than 50% we mean that the amino acid sequence comprises more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80% of amino acids. hydrophilic.
  • more than 50% we also mean that the amino acid sequence comprises at least 51%, at least 56%, at least 61%, at least 66%, at least 71%, at least 76% or at least 81% hydrophilic amino acids.
  • hydrophilic amino acids we mean the amino acids chosen from: aspartic acid (Asp / D), glutamic acid (Glu / E), arginine (Arg / R), asparagine (Asn / N), glutamine (Gin / Q ), histidine (His I H), lysine (Lys/K), serine (Ser/S) and threonine (Thr/T).
  • the invention relates to a method for preparing and determining a cPEP, said cPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a. a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein; b. a step of determining within this mRNA one of the nucleic acid sequences comprising two contiguous parts:
  • mRNA messenger RNA
  • a part located within a nucleic acid sequence known to be coding (/e. naturally translated, e.g. exon); vs. a determination step within this nucleic acid sequence comprising two contiguous parts of a fragment thereof, said fragment having a size of 3n nucleotides capable of being translated via the genetic code into a peptide, n being included from 4 to 70, in particular n being included from 4 to 41; d. a step of producing said peptide; summer. a comparison step:
  • a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is a cPEP capable of modulating the accumulation of said protein in a plant cell.
  • a cPEP can be produced by any type of means accessible to those skilled in the art.
  • a cPEP can be produced both by synthesis and by recombinant expression in homologous or heterologous systems.
  • the cPEP thus produced can then be introduced into a cell to modulate the accumulation of a target protein.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which, in step d., the production of said peptide is carried out by peptide synthesis or by recombinant expression.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which, in step d., the production of said peptide is carried out using an acid nucleic acid encoding said peptide introduced into a cell.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which, in step e., the production of said peptide is carried out using an acid nucleic acid encoding said peptide introduced into said plant cell or into said plant.
  • the invention relates to a method for preparing and determining a cPEP as described above, in which, in step e., said peptide is brought into contact with said plant cell or in said plant .
  • the invention relates to the method for preparing and determining a cPEP as described above, in which, in step e., said peptide is present in said plant cell or in said plant following the expression of a nucleic acid encoding said peptide in said plant cell or in said plant.
  • another embodiment of the invention relates to the method of preparing and determining a cPEP as described above, in which, in step e., the presence of said peptide in said plant cell or in said plant results:
  • a cPEP can be used to modulate the accumulation of a protein present naturally (/e. endogenous) or not (/e. exogenous) in said plant cell or in said plant.
  • a “protein naturally present in a plant cell or in a plant” corresponds to an endogenous protein encoded by a gene present on the genome of the plant cell or the plant without the need for direct or indirect intervention by 'a human.
  • a “protein which is not naturally present in a plant cell or in a plant” corresponds to an exogenous protein encoded by a nucleic acid sequence present on the genome of the plant cell or plant which required the intervention of a human being and the use of means known to those skilled in the art.
  • a nucleic acid sequence may come from the same plant species or from a different plant species.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is of endogenous origin in said plant cells or said plants used in step e.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is of exogenous origin in said plant cells or said plants used in step e., said plant cells or said plants used in step e. then comprising a nucleic acid sequence allowing the expression of said protein.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blot, measurement of enzyme activity, mass spectrometry and translational fusion.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
  • cPEPs makes it possible to modify the phenotypes of a plant visible on a macroscopic scale. It is therefore entirely possible to use the latter to affirm (or refute) that the peptide determined in steps a., b. and c., and optionally produced in step d. is a cPEP (or not). This is also what the so-called phenotypic comparison alternative implemented in step e allows.
  • the term "plant” refers generally: to a set of plant cells organized in all or part of a plant whatever its stage of development (including the plant in the form of seed or young grows); to one or more organs of the plant (such as leaves, roots, stem, flowers); to one or more cells of the plant; or even a mass of plant cells ⁇ eg a callus).
  • the term "phenotype" designates in a non-limiting manner the characters visible on a macroscopic scale such as the number of lateral roots, the number of leaves, the size of the stem, the duration of flowering and the resistance to stress.
  • the invention therefore relates to the method for preparing and determining a cPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • a protein is “involved in a phenotype” if a modification of the accumulation thereof is associated with a modification of said phenotype.
  • a protein is involved in a phenotype if it intervenes in the character(s) corresponding to said phenotype.
  • an object of the invention is the process for preparing and determining a cPEP as described above, in which the phenotype observed in step e. is chosen from: the size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • the invention relates to the method for preparing and determining a cPEP as described above, said cPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, and in which said plant cell (/e.
  • Alopecurus myosuroides Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said plant cells or said plants used in step e. belong to: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora , Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) is a cell of an algae.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said plant cells or said plants used in step e belong to an alga.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene chosen from: Aae15 (Acyl-activating enzyme 15), Aae16 (AMP -dependent synthetase and ligase family protein), Abcg11 (White-brown complex-like protein), Abdcg34 (ABC transporter G family member 34), Acc1 (Acetyl-CoA Carboxylase), Agb1 (GTP binding protein beta 1), Als (Acetolactate synthase (chloroplastic)), Anac076 (NAC domain-containing protein 76), Apg9 (Autophagy 9), Arlbl (GTP-binding protein 1), Arr1 (Two-component response regulator ARR1), Arr5 (Two-component response regulator ARR5) , Arr6 (Two-component response regulator ARR6), At59 (Pedate lyase family protein), Bak1 (Bracoactivating
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene chosen from: Cpk3, Dell and Nsp1.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by the Cpk3 gene.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by the Dell gene.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by the Nsp1 gene.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity. , preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NO: 1 (ORF of the Aae15 protein, A. thaliana), SEQ ID NO: 2 (ORF of the Aae16 protein, A. thaliana), SEQ ID NO: 3 (ORF of the abcg11 protein, A. thaliana), SEQ ID NO: 4 (ORF of the Abdcg34 protein, A. thaliana), SEQ ID NO: 5 (ORF of the Acc1 protein, A .
  • SEQ ID NO: 14 ORF of the protein At59, A. thaliana), SEQ ID NO: 15 (ORF of the Bak1 protein, A. thaliana), SEQ ID NO: 16 (ORF of the Bccpl protein, A. thaliana), SEQ ID NO: 17 (ORF of the Bccp2 protein, A. thaliana), SEQ ID NO: 18 (ORF of the Bri1 protein, A. thaliana), SEQ ID NO: 19 (ORF of the Bzo2h3 protein, A. thaliana), SEQ ID NO: 20 (ORF of the Cesa6 protein, A. thaliana), SEQ ID NO: 21 (ORF of the Cipk3 protein, A.
  • SEQ ID NO: 22 ORF of the Cks1 protein, A thaliana
  • SEQ ID NO: 23 ORF of the Cobl8 protein, A thaliana
  • SEQ ID NO: 24 ORF of the Coi1 protein, A. thaliana
  • SEQ ID NO: 25 ORF of the Coil protein, A. thaliana
  • SEQ ID NO: 26 ORF of the Cpk3 protein, A. thaliana
  • SEQ ID NO: 27 ORF of the Cpk3 protein, A. hypochondriacus
  • SEQ ID NO: 28 ORF of the Cpk3 protein, B. distachyori
  • SEQ ID NO: 29 ORF of the Cpk3 protein, B.
  • SEQ ID NO: 30 ORF of the Cpk3 protein, G. max
  • SEQ ID NO: 31 ORF of the Cpk3 protein, G. max
  • SEQ ID NO: 32 ORF of the Cpk3 protein, G. max
  • SEQ ID NO: 33 ORF of the Cpk3 protein, G. max
  • SEQ ID NO: 34 ORF of the Cpk3 protein, O. sativa
  • SEQ ID NO: 35 ORF of the Cpk3 protein, O. sativa
  • SEQ ID NO: 36 ORF of the Cpk3 protein, S. lycopersicum
  • SEQ ID NO: 37 ORF of the Cpk3 protein, Z.
  • SEQ ID NO: 38 ORF of the Cpk3 protein, Z. mays
  • SEQ ID NO: 39 ORF of the Cpk3 protein, Z. mays
  • SEQ ID NO: 40 ORF of the Cpk3 protein, B. rapa
  • SEQ ID NO: 41 ORF of the Cpk3 protein, B. rapa
  • SEQ ID NO: 42 ORF of the Cpk3 protein, H. vulgare
  • SEQ ID NO: 43 ORF of the Cpk3 protein, H. vulgare
  • SEQ ID NO: 44 ORF of the Cpk3 protein, S. tuberosum
  • SEQ ID NO: 45 ORF of the Cpk3 protein, A.
  • SEQ ID NO: 46 ORF of the Cpk3 protein, M. truncatula
  • SEQ ID NO: 47 ORF of the Cpk3 protein, M. truncatula
  • SEQ ID NO: 48 ORF of the Cpk3 protein, T. aestivum
  • SEQ ID NO: 49 ORF of the Cpk3 protein, T. aestivum
  • SEQ ID NO: 50 ORF of the Cpk3 protein, T. aestivum
  • SEQ ID NO: 51 ORF of the Cpk3 protein, T. aestivum
  • SEQ ID NO: 52 ORF of the Cpk3 protein, L.
  • SEQ ID NO: 53 ORF of the Cpk3 protein, L. perenne
  • SEQ ID NO: 54 ORF of the Cpk3 protein, L . perenne
  • SEQ ID NO: 55 ORF of the Cpk3 protein, L. perenne
  • SEQ ID NO: 56 ORF of the Crk34 protein, A. thaliana
  • SEQ ID NO: 57 ORF of the Cyp705a18 protein, A. thaliana
  • SEQ ID NO: 58 ORF of the Cyp71b26 protein, A. thaliana
  • SEQ ID NO: 59 ORF of the Cyp78a8 protein, A. thaliana
  • SEQ ID NO: 60 ORF of the Cyp97b3 protein , A.
  • SEQ ID NO: 61 ORF of the Dell protein, A. thaliana
  • SEQ ID NO: 62 ORF of the Dell protein, A. thaliana
  • SEQ ID NO: 63 ORF of the protein Dell, A. hypochondriacus
  • SEQ ID NO: 64 ORF of the Dell protein, B. distachyon
  • SEQ ID NO: 65 ORF of the Dell protein, G. max
  • SEQ ID NO: 66 ORF of the Dell protein, G. max
  • SEQ ID NO: 67 ORF of the Dell protein, O. sativa
  • SEQ ID NO: 68 ORF of the Dell protein, S.
  • SEQ ID NO: 69 ORF of the Dell protein, Z. mays
  • SEQ ID NO: 70 ORF of the Dell protein, B. rapa
  • SEQ ID NO: 71 ORF of the Dell protein, H. vulgare
  • SEQ ID NO: 72 ORF of the Dell protein, S. tuberosum
  • SEQ ID NO: 73 ORF of the Dell protein, M. truncatula
  • SEQ ID NO: 74 ORF of the Dell protein, T. aestivum
  • SEQ ID NO: 75 ORF of the Dell protein, T. aestivum
  • SEQ ID NO: 76 ORF of the Dell protein, T.
  • SEQ ID NO: 77 ORF of the Dell protein, T. aestivum
  • SEQ ID NO: 78 ORF of the Dell protein, L. perenne
  • SEQ ID NO: 79 ORF of the Dell protein, L. perenne
  • SEQ ID NO: 80 ORF of the Dur3 protein, A thaliana
  • SEQ ID NO: 81 ORF of the Ein2 protein, A. thaliana
  • SEQ ID NO: 82 ORF of the Emb175 protein, A. thaliana
  • SEQ ID NO: 83 ORF of the Emb2726 protein, A. thaliana
  • SEQ ID NO: 84 ORF of the Emb9 protein, A.
  • SEQ ID NO: 85 ORF of the Epsps protein, A. thaliana
  • SEQ ID NO: 86 ORF of the Fnr1 protein, A. thaliana
  • SEQ ID NO: 87 ORF of the Fve protein, A. thaliana
  • SEQ ID NO: 88 ORF of the Ga2ox7 protein, A. thaliana
  • SEQ ID NO: 89 ORF of the Gape protein, N. benthamiana
  • SEQ ID NO: 90 ORF of the Gcn2 protein, A. thaliana
  • SEQ ID NO: 91 ORF of the Gdi2 protein, A. thaliana
  • SEQ ID NO: 92 ORF of the Gln2 protein, A.
  • SEQ ID NO: 109 ORF of the Nsp1 protein, M. truncatula
  • SEQ ID NO: 110 ORF of the Nsp1 protein, A. thaliana
  • SEQ ID NO: 111 ORF of the Nsp1 protein, B. distachyon
  • SEQ ID NO: 112 ORF of the Nsp1 protein, G. max
  • SEQ ID NO: 113 ORF of the Nsp1 protein, G. max
  • SEQ ID NO: 114 ORF of the Nsp1 protein, O. sativa
  • SEQ ID NO: 115 ORF of the Nsp1 protein, S.
  • SEQ ID NO: 116 ORF of the Nsp1 protein, S. lycopersicum
  • SEQ ID NO: 117 ORF of the Nsp1 protein, Z. mays
  • SEQ ID NO: 118 ORF of the Nsp1 protein, Z. mays
  • SEQ ID NO: 119 ORF of the Nsp1 protein, Z. mays
  • SEQ ID NO: 120 ORF of the Nsp1 protein, Z. mays
  • SEQ ID NO: 121 ORF of the Nsp1 protein, B. rapa
  • SEQ ID NO: 122 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 123 ORF of the Nsp1 protein, H.
  • SEQ ID NO: 124 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 125 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 126 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 127 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 128 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 129 ORF of the Nsp1 protein, S. tuberosum
  • SEQ ID NO: 130 ORF of the Nsp1 protein, S. tuberosum
  • SEQ ID NO: 131 ORF of the Nsp1 protein, T.
  • SEQ ID NO: 132 ORF of the Nsp1 protein, T. aestivum
  • SEQ ID NO: 133 ORF of the Nsp1 protein, L. perenne
  • SEQ ID NO: 134 ORF of the Nsp1 protein, L. perenne
  • SEQ ID NO: 135 ORF of the Pds protein, A. thaliana
  • SEQ ID NO: 136 ORF of the Pen3 protein, A. thaliana
  • SEQ ID NO: 137 ORF of the Phyb protein, A. thaliana
  • SEQ ID NO: 138 ORF of the Pif3 protein, A.
  • SEQ ID NO: 139 ORF of the Pizza protein, A thaliana
  • SEQ ID NO: 140 ORF of the Ppoxl protein, A thaliana
  • SEQ ID NO: 141 ORF of the Ppox2 protein, A. thaliana
  • SEQ ID NO: 142 ORF of the Prp39 protein, A. thaliana
  • SEQ ID NO: 143 ORF of the PsbA protein, A. thaliana
  • SEQ ID NO: 144 ORF of the Pskrl protein, A. thaliana
  • SEQ ID NO: 145 ORF of the Rd21 protein, A. thaliana
  • SEQ ID NO: 146 ORF of the Ringl protein, A.
  • SEQ ID NO: 147 ORF of the Rosi protein, A. thaliana
  • SEQ ID NO: 148 ORF of the Rpt4a protein, A. thaliana
  • SEQ ID NO: 149 ORF of the Sfr6 protein, A. thaliana
  • SEQ ID NO: 150 ORF of the Shr protein, A. thaliana
  • SEQ ID NO: 151 ORF of the Shy2 protein, A. thaliana
  • SEQ ID NO: 152 ORF of the Ski protein, M. truncatula
  • SEQ ID NO: 153 ORF of the Sps1 protein, A. thaliana
  • SEQ ID NO: 154 ORF of the Spt protein, A.
  • SEQ ID NO: 155 ORF of the Stn8 protein, A. thaliana
  • SEQ ID NO: 156 ORF of the Tap46 protein, A. thaliana
  • SEQ ID NO: 157 ORF of the Topp6 protein, A. thaliana
  • SEQ ID NO: 158 ORF of the TubB6 protein, A. thaliana
  • SEQ ID NO: 159 ORF of the TubB8 protein, A. thaliana
  • SEQ ID NO: 160 ORF of the llbala protein, A. thaliana
  • SEQ ID NO: 161 ORF of the Vim3 protein, A. thaliana
  • SEQ ID NO: 381 ORF of the Sgr1 protein, A.
  • SEQ ID NO: 382 ORF of the Abi5 protein, A. thaliana
  • SEQ ID NO: 383 ORF of the Hsp101 protein, A. thaliana
  • SEQ ID NO: 384 ORF of the Rh10 protein, M. truncatula
  • SEQ ID NO: 385 ORF of the Wus protein, A. thaliana
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity. , preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1 ).
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • percentage of identity between two nucleic acid (or amino acid) sequences, we mean a percentage of nucleotides (or amino acid residues) identical between the two sequences to be compared, obtained after the best alignment. . This percentage is purely statistical and the differences between the two sequences are randomly distributed over the entire length of the sequences.
  • the best alignment is the alignment for which the percentage of identity between the two sequences to be compared, as calculated below, is the highest.
  • Sequence comparisons between two nucleic acid (or amino acid) sequences are traditionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out per segment or per comparison window to identify and compare the regions local sequence similarity.
  • the optimal alignment of sequences for comparison can be carried out manually or by means of algorithms and software available to those skilled in the art, for example, the BLAST platform or the MatGat program (Campanella, Bitincka and Smalley , 2003).
  • the percentage identity between two sequences is determined by comparing these two optimally aligned sequences by comparison window in which the region of the sequence to be compared may include additions or deletions relative to the reference sequence for optimal alignment between these two sequences. Percent identity is calculated by determining the number of identical positions for which the nucleotide (or amino acid) is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100.
  • sequences presenting “at least 80% identity” with a reference sequence may in particular present at least 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% d identity with said reference sequence.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs : 1 to 161 and 381 to 385.
  • the invention also relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a sequence of nucleic acids chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NO: 162 (cPEPcpk3), SEQ ID NO: 163 (cPEPdcll), SEQ ID NO: 164 (cPEPnsp1_3), SEQ ID NO: 165 (cPEPnsp1_1), SEQ ID NO: 166 (cPEPnsp1_2), SEQ ID NO: 167 (cPEPnsp1_4), SEQ ID NO: 168 (cPEPnsp1_5 ), SEQ ID NO: 169 (cPEPnspl 5aa), SEQ ID NO: 170 (cPEPnspl 20aa), SEQ ID NO: 171 (cPEPnspl 30aa), SEQ ID NO: 172 (cPEPnspl 40aa), SEQ ID NO: 173 (cPEPnsp
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NOs : 164 to 174.
  • the invention relates to the method for preparing and determining a cPEP as described above, in which the sequence of said peptide is the sequence SEQ ID NO: 162.
  • the invention relates the process for preparing and determining a cPEP as described above, in which the sequence of said peptide is the sequence: SEQ ID NO: 163.
  • the invention relates to the process for preparing and determining a cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NOs: 164 to 174.
  • the subject of the invention above is a cPEP as obtained by implementing the method as described above.
  • the invention also relates to a cPEP, of 4 to 70 amino acids, in particular of 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment (not naturally translated) of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to cPEP as previously described, said fragment having a size of 3n nucleotides, n being included: from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
  • the invention relates to cPEP as described above, said cPEP comprising s, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids .
  • the invention relates to cPEP as described above, said cPEP comprising from 4 to 41 amino acids.
  • the invention relates to cPEP as described above, said cPEP comprising from 5 to 40 amino acids.
  • the invention relates to cPEP as described above, said cPEP comprising from 7 to 20 amino acids.
  • the invention also relates to cPEP as described above, said cPEP comprising 8 to 15 amino acids.
  • the invention relates to cPEP as described above, in which the size of said cPEP is less than that of said protein.
  • the invention relates to cPEP as described above, said fragment lacking: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention therefore relates to cPEP as described above, said fragment comprising an AUG initiator codon encoding an initiator methionine and being devoid of a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention also relates to cPEP as described above, said fragment being devoid of an AUG initiator codon encoding an initiator methionine and comprising a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention relates to cPEP as described above, said fragment lacking: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and in which said fragment is chosen: either in the same reading frame as the open reading frame encoding said protein; either in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein.
  • the invention relates to cPEP as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the naturally translated nucleic acid sequence. in said plant cell.
  • the invention relates to cPEP as described previously, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
  • the invention relates to cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or two nucleotides in 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the subject of the invention is a cPEP, of 4 to 70 amino acids, in particular of 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence of an mRNA of a protein, said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/e. non translated naturally, e.g. 3'IITR or 5'IITR); and a part located within a nucleic acid sequence deemed to be coding (/e. naturally translated, e.g.
  • said fragment having a size of 3n nucleotides, n being included from 4 to 70, in particular n being included from 4 to 41, and said cPEP being capable of modulating the accumulation of said protein in a plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to cPEP as described above, wherein said cPEP is capable of increasing the accumulation of said protein in said plant cell. In one embodiment, the invention relates to cPEP as described above, in which said cPEP is capable of reducing the accumulation of said protein in said plant cell.
  • the invention relates to cPEP as described above, in which said cPEP is a synthetic peptide.
  • the invention relates to cPEP as described above, in which said cPEP is an isolated peptide.
  • the invention relates to cPEP as described above, in which said cPEP is a recombinant peptide.
  • the invention relates to cPEP as described above, said cPEP being a hydrophobic peptide or a hydrophilic peptide.
  • the invention relates to cPEP as described above, in which said protein is naturally present in said plant cell.
  • the invention relates to cPEP as described above, in which said protein is not naturally present in said plant cell.
  • the invention relates to cPEP as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell.
  • the invention relates to cPEP as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
  • the invention relates to cPEP as described above, in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • a plant species chosen from
  • the invention relates to cPEP as described above, in which said plant cell is a cell of an alga.
  • the invention relates to cPEP as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acd, Agb1, Als, Anac076, Apg9, Arlbl, Arr1 , Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9 , Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lr
  • the invention relates to cPEP as described above, in which said protein is encoded by a gene chosen from the genes: Cpk3, Dell and Nsp1.
  • the invention relates to cPEP as described above, in which said protein is encoded by the Cpk3 gene.
  • the invention relates to cPEP as described above, in which said protein is encoded by the Dell gene.
  • the invention relates to cPEP as described above, in which said protein is encoded by the Nsp1 gene.
  • the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention relates to the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention also relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention also relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention also relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention relates in particular to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NOs: 162 to 206, 404 and 406 to 417.
  • the invention relates to cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NOs: 164 to 174.
  • the invention the cPEP as described previously, in which the sequence of said peptide is the sequence SEQ ID NO: 162.
  • the invention relates to the cPEP as described previously, in which the sequence of said peptide is the sequence: SEQ ID NO: 163.
  • the invention relates to cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NOs: 164 to 174.
  • the invention relates to cPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • plant phenotype chosen from: size, shape, surface area, volume, mass and number leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration
  • a cPEP may be fused or linked to one or more molecules facilitating entry of the cPEP into the cell.
  • penetrating peptides Numata, K., et al. Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Sci Rep 8 , 10966 (2016).
  • palmitic acid By “penetrating peptide” (hereinafter CPP), we mean small peptides penetrating cellular lipid bilayers or destabilizing cell membranes. CPPs can be classified into three groups: cationic, amphipathic and hydrophobic.
  • cationic CPPs contain many positively charged amino acids, such as lysine (Lys) and arginine (Arg); amphipathic CPPs are generally composed of an alternating sequence of polar and nonpolar amino acids; and hydrophobic CPPs consist of nonpolar amino acids with relatively low net charges.
  • the invention relates to cPEP as described above, said cPEP being fused to a peptide facilitating its entry into the plant cell.
  • the invention relates to cPEP as described above, said cPEP being fused to a penetrating peptide.
  • the invention relates to cPEP as described above, said cPEP being fused at the N-terminal end or at the C-terminal end with said peptide facilitating its entry into the plant cell.
  • the invention relates to cPEP as described above, said cPEP being fused at the N-terminus or at the C-terminus with said penetrating peptide.
  • the invention relates to cPEP as described above, said cPEP being fused with: the TAT peptide (SEQ ID NO: 380); penetratin; a polyhistidine peptide (in particular a peptide of at least 4 histidine residues); or a polyarginine peptide (in particular a peptide of 4 arginine residues).
  • the invention relates to cPEP as described above, said cPEP being linked to one or more palmitic acid molecules.
  • the invention relates to cPEP as described above, said cPEP being linked at the N-terminus or at the C-terminus to one or more palmitic acid molecules.
  • the quantity of cPEP necessary to modulate the accumulation of a protein can vary depending on whether or not the cPEP is modified with one of the molecules facilitating its cellular penetration.
  • the invention above relates to a nucleic acid encoding a cPEP as described above.
  • the invention also relates to a nucleic acid of 3n nucleotides, which nucleic acid corresponds to a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA.
  • the invention relates to the nucleic acid described above, said fragment lacking: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention therefore relates to the nucleic acid as described above, said fragment comprising an AUG initiator codon encoding an initiator methionine and being devoid of a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention also relates to the nucleic acid as described above, said fragment being devoid of an AUG initiator codon encoding an initiator methionine and comprising a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention relates to the nucleic acid described above, said fragment lacking: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the nucleic acid sequence. naturally translated in said plant cell.
  • the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
  • the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally occurring nucleic acid sequence. translated into said plant cell.
  • the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in the 3' (or by two nucleotides in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5' ) relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3 ' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to a nucleic acid of 3n nucleotides, which nucleic acid corresponds to a fragment of a nucleic acid sequence of an mRNA of a protein, said nucleic acid sequence comprising two parts contiguous: a part located within a nucleic acid sequence deemed non-coding (/e. not naturally translated, eg 3'UTR or 5'IITR); and a part located within a nucleic acid sequence known to be coding (/e. naturally translated, eg exon).
  • the invention relates to the nucleic acid as described above, where n is comprised: from 4 to 70; from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
  • the above invention relates to a composition
  • a composition comprising a cPEP as described above as an active substance.
  • the invention relates to a composition
  • a composition comprising a cPEP as active substance, said cPEP:
  • the invention relates to the composition as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention therefore relates to the composition as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention also relates to the composition as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention relates to the composition as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the naturally translated nucleic acid sequence. in said plant cell.
  • the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
  • the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relation to the open reading frame of the acid sequence nucleic acids naturally translated in said plant cell.
  • the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to a composition
  • a composition comprising a cPEP as active substance, said cPEP:
  • nucleic acid sequence comprising two contiguous parts:
  • ⁇ a part located within a nucleic acid sequence known to be coding (/e. naturally translated, e.g. exon);
  • the invention relates to the composition as described above, in which said cPEP is at a concentration comprised from 10'9 M to 10'3 M.
  • said composition of the invention does not exist in the natural state and this is all the more true since such a concentration of cPEP cannot exist within a plant cell.
  • concentration comprised from 10' 9 M to 10' 3 M it is understood that the concentration of cPEP can be comprised from 10' 9 to 10' 4 M, from 10' 8 to 10' 4 M, from 10' 9 to 10' 5 M, from 10' 8 to 10' 5 M, as it can be comprised from 5 pM to 500 pM, from 30 pM to 70 pM, or even be 50 pM.
  • the invention relates to the composition as described above, in which said cPEP is at a concentration comprised from 10' 9 to 10' 4 M, from 10' 8 to 10' 4 M, from 10' 9 to 10' 5 M or from 10'8 to 10'5 M.
  • the invention relates to the composition as described above, in which said cPEP is at a concentration of from 5 pM to 500 pM or from 30 pM to 70 pM.
  • the invention relates to the composition such as described previously, in which said cPEP is at a concentration of 50 pM. In a non-limiting manner, this concentration can also be 10' 9 M, 10' 8 M, 10' 7 M, 10' 6 M, 10' 5 M or IO' 4 M.
  • the invention also relates to the composition comprising a cPEP as active substance, said cPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, of which the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA ; being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein; and being in particular at a concentration of 5 pM to 500 pM or from 30 pM to 70 pM, or being in particular at a concentration of 50 pM.
  • composition comprising a cPEP comprises at least one cPEP. That is to say, a mixture of cPEPs is possible, said cPEPs being able to target the same protein or several proteins depending on the nucleic acid fragment from which they come.
  • concentrations concern either the mixture of cPEPs as such, or each of the cPEPs of said mixture, said cPEPs being able to be at the same concentration or being able to be at different concentrations among those cited above.
  • the invention relates to the composition as described above, said composition being a phytopharmaceutical composition, a herbicidal composition or a coating composition, in particular said coating composition further comprising at least one fixing agent .
  • the invention relates to the composition as described above, said composition being a phytopharmaceutical composition.
  • the invention relates to the composition as described above, said composition being a herbicidal composition.
  • the invention relates to the composition as described above, said composition being a coating composition.
  • the invention relates to the composition as described above, said composition being a coating composition further comprising at least one fixing agent.
  • the invention relates to the composition as described above, said composition further comprising at least one solvent.
  • said solvent is chosen from: acetone, acetonitrile, acetic acid, formic acid, dimethyl adipate, benzyl acetate, bi-butyl carbonate, dimethyl sulfoxide (DMSO), water, dimethyl glutarate, ammonium hydroxide, iso-butanol, iso-propanol, diethyl hexyl lactate, light aromatic solvent naphtha, heavy aromatic solvent naphtha, diethyl succinate and their mixtures (eg mixture [water; acetic acid]; [acetonitrile; acetic acid], [water, acetonitrile; acetic acid], [water; DMSO], [water; acetonitrile] or [water; ammonium hydroxide]) .
  • DMSO dimethyl sulfoxide
  • solubility properties of cPEPs are determined in particular by their amino acid composition.
  • Hydrophilic cPEPs can be solubilized and packaged in aqueous solutions, such as water.
  • Hydrophobic cPEPs can be solubilized and packaged in solvents, such as organic solvents.
  • organic solvents are non-toxic solvents for plants in small quantities, that is to say they have no deleterious effect on the development of the plant.
  • the organic solvents may be those mentioned above and in particular chosen from acetonitrile and acetic acid.
  • cPEPs can also be solubilized and packaged in solvent mixtures, such as, for example, an organic solvent mixture [acetonitrile; acetic acid], a mixture [water; DMSO] in a volume:volume ratio of 99:1 to 1:99, a mixture [water; acetonitrile] in a volume:volume ratio of 99:1 to 1:99 or a mixture [water; ammonium hydroxide] in a volume:volume ratio of 99:1 to 99.9:0.1.
  • solvent mixtures such as, for example, an organic solvent mixture [acetonitrile; acetic acid], a mixture [water; DMSO] in a volume:volume ratio of 99:1 to 1:99, a mixture [water; acetonitrile] in a volume:volume ratio of 99:1 to 1:99 or a mixture [water; ammonium hydroxide] in a volume:volume ratio of 99:1 to 99.9:0.1.
  • the cPEPs can also be solubil
  • the invention relates to the composition as described above, said composition further comprising at least one diluent.
  • the invention relates to the composition as described above, said composition further comprising at least one adjuvant.
  • the invention relates to the composition as described above, said composition further comprising at least one fixing agent.
  • fixing agent is meant a chemical or natural agent which makes it possible to stick the composition of the invention to a plant seed so as to coat said seed with vegetal.
  • a substance making it possible to apply and hold the active substance(s) on the grain.
  • fixing agents available, we find in particular carboxymethyl cellulose (CMC) and gum arabic.
  • a fixing agent may include organic solvents, water, dispersants, emulgators, surfactants, wetting agents and dyes.
  • the invention relates to the composition as described above, said composition further comprising at least one plant nutrient.
  • the invention relates to the composition as described above, said composition further comprising at least one fixing agent and at least one plant nutrient.
  • plant nutrient we mean an element assimilated by the plant to enable its development.
  • a plant nutrient can be chosen from: nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, manganese, iron, copper, boron, zinc, molybdenum and their mixtures.
  • a coated seed comprising a plant seed, said plant seed being coated with a coating composition as described above.
  • the coating can be carried out according to the processes conventionally used in the food industry and can be obtained by using a material capable of disintegrating in a solvent or in the earth, such as a binder or clay.
  • the coating can be used to confer particular properties to a seed in combination with a cPEP, such as improved growth or resistance to certain biotic or abiotic stresses.
  • the invention relates to the coated seed as described above, in which said plant seed has a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacu
  • the invention relates to coated seed as described above, said seed being treated by soaking in a composition containing a cPEP. During soaking, the seed is then totally or partially immersed in a composition containing a cPEP.
  • the above invention relates to a use of a cPEP as a phytosanitary agent to modulate the accumulation of a protein in a plant cell
  • said cPEP having a size ranging from 4 to 70 amino acids , in particular from 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the framework reading open of said protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the use of a cPEP as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention therefore relates to the use of a cPEP as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG , UGA and UAA.
  • the invention also relates to the use of a cPEP as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention relates to the use of a cPEP as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention relates to the use of a cPEP as described previously, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the sequence d nucleic acids naturally translated in said plant cell.
  • the invention relates to the use of a cPEP as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
  • the invention relates to the use of a cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the sequence of nucleic acids naturally translated in said plant cell.
  • the invention relates to the use of a cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the use of a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in 3' (or by one nucleotide in 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the use of a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the use of a cPEP as a phytosanitary agent to modulate the accumulation of a protein in a plant cell, said cPEP having a size of 4 to 70 amino acids, in particular 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a naturally occurring nucleic acid sequence translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being between 4 and 70, in particular n being between 4 and 41, and said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/e.
  • cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the use of a cPEP as described above to increase the accumulation of said protein in the plant cell.
  • the presence of cPEP causes the quantity of said protein in the treated plant cell to be greater than that in an untreated plant cell.
  • the invention relates to the use of a cPEP as described above to reduce (inhibit) the accumulation of said protein in the plant cell.
  • the presence of cPEP causes the quantity of said protein in the treated plant cell to be lower than that in an untreated plant cell.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is produced outside of said plant cell before being introduced into said plant cell.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is a synthetic peptide.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is an isolated peptide.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is a recombinant peptide.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is a hydrophobic peptide or a hydrophilic peptide.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is introduced into said plant cell in the form of a nucleic acid encoding said cPEP.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is introduced into said plant cell in the form of a nucleic acid encoding said cPEP and comprising the means of expressing it.
  • the invention relates to the use of a cPEP as described above, in which said protein is naturally present in said plant cell.
  • the invention relates to the use of a cPEP as described above, in which said protein is not naturally present in said plant cell.
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell.
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
  • the invention relates to the use of a cPEP as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blotting, measurement of enzymatic activity, mass spectrometry and translational fusion.
  • the invention relates to the use of a cPEP as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
  • the invention relates to the use of a cPEP as described above, in which said cPEP has a size comprised from 4 to 41 amino acids, from 5 to 40 amino acids, from 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids.
  • said cPEP has a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • the invention relates to the use of a cPEP as described above, in which said plant cell belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii , Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amarant
  • the invention relates to the use of a cPEP as described above, in which said plant cell is a cell of an alga.
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Aribl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, L
  • the invention relates to the use of a cPEP as described previously, said cPEP having a size of 4 to 70 amino acids, in particular 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a non-naturally translated fragment of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open frame of reading of said protein encoded by said mRNA, said non-naturally translated fragment being devoid of the AUG initiator codon encoding an initiator methionine and/or a STOP codon chosen from the codons: UAG, UGA and UAA, and being chosen either in the same reading frame that the open reading frame encoding said protein, is in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein, and said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the m
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a sequence of nucleic acids chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention also relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the use of a cPEP as described above, in which said cPEP is chosen from the sequences: SEQ ID NOs: 162 to 206, 404 and 406 to 417.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is chosen from the sequences: SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NOs: 164 to 174.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is of sequence SEQ ID NO: 162.
  • the invention relates to the use of a cPEP as described previously, in which said cPEP is of sequence: SEQ ID NO: 163.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is chosen from the sequences: SEQ ID NOs: 164 to 174 .
  • the invention relates to the use of a cPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume , the mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • plant phenotype chosen from: size, shape, surface area, volume , the mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (
  • the invention relates to the use of a cPEP as described above, to modulate the accumulation of a recombinant protein whose nucleic acid sequence which encodes it corresponds to the fusion of the sequences of nucleic acids of two distinct genes.
  • the coding sequence of at least one of the two genes is that of a reporter gene, for example a gene encoding a fluorescent protein (such as GFP) or a protein allowing resistance of the plant to a compound.
  • a reporter gene for example a gene encoding a fluorescent protein (such as GFP) or a protein allowing resistance of the plant to a compound.
  • the invention relates to the use of a cPEP as described above, to modulate the accumulation of a recombinant protein whose nucleic acid sequence which encodes it corresponds to the fusion: of a nucleic acid sequence deemed non-coding of a first gene; and a coding nucleic acid sequence of a second gene, the sequence of said cPEP corresponding to the translation via the genetic code of a fragment of the nucleic acid sequence deemed non-coding of the first gene.
  • the above invention relates to a method of modulating the accumulation of a protein in a plant cell comprising a step of introducing: a cPEP; or a nucleic acid encoding said cPEP and the means of expressing it, in said plant cell, the introduction of said cPEP leading to a modulation of the quantity of said protein in said plant cell, said cPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the method as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention therefore relates to the method as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention also relates to the method as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention relates to the method as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the naturally translated nucleic acid sequence. in said plant cell.
  • the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
  • the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a different reading frame. of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or two nucleotides in 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to a method for modulating the accumulation of a protein in a plant cell comprising a step of introducing: a cPEP; or a nucleic acid encoding said cPEP and the means of expressing it, in said plant cell, the introduction of said cPEP leading to a modulation of the quantity of said protein in said plant cell, said cPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, and said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/e.
  • cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the method as described above, said method allowing: to promote the development of a plant; or slow down or prevent the development of a plant.
  • the invention relates to the process as described above, said process making it possible to promote the development of a plant.
  • the invention relates to the process as described above, said process making it possible to slow down or prevent the development of a plant.
  • the invention relates to the method as described above for increasing the accumulation of said protein in the plant cell.
  • the presence of cPEP causes the quantity of said protein in the treated plant cell to be greater than that in an untreated plant cell.
  • the invention relates to the method as described above for reducing (inhibiting) the accumulation of said protein in the plant cell.
  • the presence of cPEP causes the quantity of said protein in the treated plant cell to be lower than that in an untreated plant cell.
  • the invention relates to the method as described above, in which said cPEP is produced outside of said plant cell before being introduced into said plant cell.
  • the invention relates to the method as described above, in which said cPEP is a synthetic peptide.
  • the invention relates to the method as described above, in which said cPEP is an isolated peptide.
  • the invention relates to the method as described above, in which said cPEP is a recombinant peptide.
  • the invention relates to the method as described above, in which said cPEP being a hydrophobic peptide or a hydrophilic peptide.
  • the invention relates to the method as described above, in which said cPEP is introduced into said plant cell in the form of a nucleic acid encoding said cPEP.
  • the invention relates to the method as described above, in which said cPEP is introduced into said plant cell in the form of a nucleic acid encoding said cPEP and comprising the means of expressing it.
  • the invention relates to the method as described above, in which said protein is naturally present in said plant cell.
  • the invention relates to the method as described above, in which said protein is not naturally present in said plant cell.
  • the invention relates to the method as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell.
  • the invention relates to the method as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
  • the invention relates to the method as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blotting, measurement of enzymatic activity , mass spectrometry and translational fusion.
  • the invention relates to the method as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
  • the invention relates to the method as described above, in which said cPEP has a size comprised from 4 to 41 amino acids, from 5 to 40 amino acids, from 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids.
  • said cPEP has a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
  • the invention relates to the method as described above, in which said plant cell or said plant belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata , Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean ), Gossypium raimondii, Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochon
  • the invention relates to the method as described above, in which said plant cell is an algae cell.
  • the invention relates to the method as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1 , Arr5, Arrô, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9 , Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx
  • the invention relates to the method as described above comprising a step of introducing: a cPEP; or a nucleic acid encoding said cPEP and the means of expressing it, in said plant cell, the introduction of said cPEP leading to a modulation of the quantity of said protein in said plant cell, said cPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a non-naturally translated fragment of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said non-naturally translated fragment being devoid of the AUG initiator codon encoding an initiator methionine and/or a STOP codon chosen from the codons: UAG, UGA and UAA , and being chosen either in the same reading frame as the open reading frame coding said protein, or
  • the invention relates to the method as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention relates to the method as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385. In one embodiment, the invention also relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method as described above, in which said cPEP is chosen from the sequences: SEQ ID NOs: 162 to 206, 404 and 406 to 417.
  • the invention relates to the method as described above, in which said cPEP is chosen from the sequences: SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NOs: 164 to 174.
  • the invention concerns relates to the method as described above, in which said cPEP is of sequence SEQ ID NO: 162.
  • the invention relates to the method as described previously, in which said cPEP is of sequence: SEQ ID NO: 163.
  • the invention relates to the use of a cPEP as described above, in which said cPEP is chosen from the sequences: SEQ ID NOs: 164 to 174.
  • the invention relates to the method as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • the invention relates to the method as described above, in which the introduction of said cPEP results in early bolting in said plant.
  • the invention relates to the method as described above, in which the introduction of said cPEP results in earlier flowering in said plant.
  • the invention relates to the method as described above, in which the introduction of said cPEP causes an increase in the size of the stem in said plant.
  • the invention relates to the method as described above, in which the introduction of said cPEP results in earlier growth of the stem in said plant.
  • a cPEP to the plant, e.g. via the use of the composition of the invention (see above) comprising a cPEP, to modulate the accumulation of a target protein in the plant, indicating that cPEP is taken up by the plant.
  • the invention relates to the method as described above, in which said cPEP is introduced into said plant: by watering, by spraying or by adding a fertilizer, a potting soil, a culture substrate or a support in contact with the plant, said cPEP being in particular administered to the plant in the form of a composition comprising from IO' 9 M to IO' 4 M of said cPEP; by watering, by soaking, by spraying or by adding a fertilizer, a potting soil, a growing substrate or a support in contact with the plant, said cPEP being in particular administered to a seed or a seed in the form of a composition comprising from 10'9 M to 10'4 M of said cPEP; or by means of a nucleic acid encoding said cPEP and comprising the means of expressing said cPEP, said nucleic acid being artificially introduced into the plant.
  • the invention relates to the method as defined above, in which said cPEP is artificially introduced externally into the plant, preferably by watering, by spraying or by the addition of a fertilizer, potting soil, a growing medium or an inert support. In one embodiment, the invention relates to the method as defined above, in which said cPEP is introduced by watering.
  • the invention relates to the method as defined above, in which said cPEP is introduced by spraying.
  • the invention relates to the process as defined above, in which said cPEP is introduced by the addition of a fertilizer.
  • the invention relates to the process as defined above, in which the plant is treated with a composition comprising from 10' 9 M to 10' 4 M of said cPEP, or comprising in particular 10' 9 M, 10' 8 M, 10' 7 M, 10' 6 M, 10' 5 M or 10' 4 M of said cPEP.
  • the compositions have a concentration of 10'8 M to 10'5 M for application by watering or spraying on the plant.
  • more or less concentrated compositions can be considered to treat the plant with cPEP.
  • more concentrated compositions comprising from 10' 1 M to 10' 3 M, or comprising in particular 10' 2 M of cPEP, can be used in the case where the cPEP artificially introduced externally is administered to the plant by spreading.
  • the above invention relates to a modified plant containing a cPEP, which “modified plant” corresponds to a plant into which a cPEP has been artificially introduced, in particular by watering, by spraying or via fertilizer.
  • the invention relates to the modified plant comprising a cPEP introduced exogenously, said cPEP having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said cPEP being capable of modulate the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the modified plant as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; Or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention therefore relates to the modified plant as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA .
  • the invention also relates to the modified plant as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention relates to the modified plant as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the naturally occurring nucleic acid sequence. translated into said plant cell.
  • the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
  • the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally translated nucleic acid sequence. in said plant cell.
  • the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the plant modified as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides at 3' (or by one nucleotide at 5') relative to the open reading frame of the sequence of nucleic acids naturally translated in said plant cell.
  • the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in the 5') or two nucleotides in the 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to a modified plant comprising an exogenously introduced cPEP, said cPEP having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, and said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/.e. not naturally translated, e.g.
  • 3'IITR or 5'IITR and a part located within a nucleic acid sequence known to be coding (/e. naturally translated, e.g. exon); said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the modified plant as described above, said plant belonging to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacusAmaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp .
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacusA
  • the above invention relates to a transgenic plant comprising a nucleic acid encoding a cPEP and the means for expressing it, said cPEP having a size of from 4 to 70 amino acids, in particular from 4 to 41 acids. amino, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of an encoded protein by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the transgenic plant as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention therefore relates to the transgenic plant as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA .
  • the invention also relates to the transgenic plant as described above, in which said fragment lacks an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
  • the invention relates to the transgenic plant as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
  • the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
  • the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally translated nucleic acid sequence. in said plant cell.
  • the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in the 5') or two nucleotides in the 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to a transgenic plant comprising a cPEP introduced exogenously, said cPEP having a size of from 4 to 70 amino acids, in particular from 4 to 70 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, and said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/.e.
  • cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the transgenic plant as defined above, in which the sequence encoding said cPEP is shorter than the sequence of the mRNA encoding said protein.
  • the invention relates to the transgenic plant as described above, said plant belonging to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus
  • the invention relates to the transgenic plant as described above, in which the expression of said cPEP is placed under the control of a strong promoter, preferably a strong constitutive promoter such as the 35S promoter.
  • a first aspect thereof relates to a method for preparing and determining an altPEP, said altPEP: having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids ; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a.
  • a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein b. a step of determining within this mRNA the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein; vs. a step of determining within this nucleic acid sequence naturally translated in said plant cell a naturally translated fragment thereof, said fragment having a size of 3n nucleotides capable of being translated via the genetic code into a peptide , n being between 4 and 70, in particular n being between 4 and 41, and said fragment having a size smaller than that of the nucleic acid sequence naturally translated in said plant cell; d. a step of producing said peptide; summer. a comparison step:
  • a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is an altPEP capable of modulating the accumulation of said protein in a plant cell.
  • the present invention is based on the unexpected observation made by the inventors that it is possible to specifically modulate the accumulation of a protein using a particular peptide produced naturally, the sequence of which corresponds to the translation of a fragment messenger RNA (mRNA) encoding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
  • mRNA fragment messenger RNA
  • altPEP alternative peptide designates a peptide capable of specifically modulating the accumulation of a protein once introduced into a plant cell.
  • an altPEP is naturally present in a plant cell.
  • the plant cell contains the altPEP information and the means to allow its expression (/.e. START codon and STOP codon).
  • An altPEP may therefore be present in a plant cell and the quantity thereof may be modified by the artificial addition of it, in the form of a peptide or in the form of a nucleic acid encoding said peptide. , in the plant cell.
  • the specificity of altPEP towards a target protein is determined by its amino acid sequence. Indeed, the sequence of an altPEP corresponds to the translation of a fragment of the mRNA encoding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
  • the peptide sequence of an altPEP can therefore be determined from a fragment of the mRNA encoding said protein.
  • the fragment of the mRNA used to determine the sequence of altPEP can be chosen in the two other reading frames than that coding the protein whose accumulation it is desired to modulate existing on the sequence of the mRNA .
  • a fragment can be selected in reading frames +2 or +3.
  • the other two reading frames (+2 and +3) contain the information of an altPEP as it is possible that only one of the two reading frames (+2 or +3) contain the information of an altPEP.
  • reading frame designates the grouping of nucleotides constituting a sequence of nucleic acids into consecutive triplets (or codons), which follow one another without interruption or overlap.
  • altPEPs have a size of 4 to 70 amino acids, in particular of 4 to 41 amino acids, in particular a size of 5 to 40 amino acids, of 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids.
  • sequence of an altPEP corresponds to the translation of a fragment comprising 4 to 41 nucleotide triplets, in particular a fragment comprising 5 to 40 nucleotide triplets, 7 to 20 nucleotide triplets or more particularly of a fragment comprising 8 to 15 nucleotide triplets.
  • sequence of an altPEP corresponds to the translation into amino acids of a fragment of “3n” nucleotides of the mRNA of the target protein, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, in particular from 5 to 40, from 7 to 20 or more particularly from 8 to 15.
  • n is equal to 40
  • n is equal to 70
  • the altPEPs have a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said fragment has a size of 3n nucleotides, n being included: from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said peptide has a size chosen from: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 and 70 amino acids.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said altPEP has a size smaller than that of said protein (/e. that of which altPEP modulates accumulation).
  • altPEPs have the capacity to specifically modulate the accumulation of a protein without the accumulation of the corresponding mRNA being impacted.
  • the addition of an altPEP in a plant cell does not modify the quantity of mRNA allowing the expression of the protein that it has the capacity to regulate, but only the quantity of said protein.
  • the term “protein” designates a sequence of amino acids whose information is encoded by a gene present on the genome of a plant cell.
  • gene we therefore designate, in particular, the nucleic acid sequence necessary for the synthesis of said protein.
  • a gene includes more than the nucleotides encoding the amino acid sequence of the protein.
  • a gene includes the DNA sequences necessary for the synthesis of a pre-messenger (pre-mRNA), which is then processed by the cellular machinery into messenger RNA (mRNA). The latter can then be translated, via ribosomes, into a protein.
  • pre-mRNA pre-messenger
  • mRNA messenger RNA
  • pre-messenger RNA has not undergone splicing and is likely to contain introns
  • mature messenger RNA may have undergone a splice and contains only exons.
  • an altPEP capable of modulating the accumulation of a protein
  • the “modulation” of the accumulation of a protein designates either an increase in the accumulation of said protein (/e. an increase in the quantity of protein in the plant cell), or a decrease in the accumulation of said protein (/e. a decrease in the quantity of protein in the plant cell).
  • one embodiment of the invention relates to the method of preparing and determining a altPEP as described above, wherein said modulation of the accumulation of said protein induced by said altPEP is: a decrease in the accumulation of said protein; or an increase in the accumulation of said protein.
  • the increase and decrease in the accumulation of said protein can be measured and monitored using methods well known to those skilled in the art, such as the coupling of the protein to a marker via the use of cassettes. particular expression, or a Western blot.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which in step e., the quantity of protein in the presence of said peptide is greater than the quantity of protein in the absence of said peptide.
  • step e. the quantity of protein in the presence of said peptide is greater than the quantity of protein in the absence of said peptide.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which in step e., the quantity of protein in the presence of said peptide is less than the quantity of protein in the absence of said peptide.
  • step e. the quantity of protein in the presence of said peptide is less than the quantity of protein in the absence of said peptide.
  • the fragment of the mRNA encoding said altPEP is located within the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein, and said fragment as such can also be translated naturally in said plant cell.
  • the existence of an altPEP within said plant cell is therefore natural and originates either from cellular machinery or from human action. For this, it is possible either to artificially introduce said altPEP as such, or to introduce an expression cassette comprising the nucleic acid sequence encoding said altPEP and the means of expressing it in said plant cell.
  • the nucleic acid sequence naturally translated in said plant cell which comprises a fragment carrying the information of an altPEP, is a region of the mRNA described as “coding”, that is to say that it corresponds to a region of the mRNA which codes all or part of the functional protein.
  • This sequence therefore corresponds to the main open reading frame (/.e. the +1), which codes the protein whose accumulation we want to modulate.
  • open reading frame and “ORF” (open reading frame) are equivalent, and can be used interchangeably. They correspond to a sequence of nucleotides (nucleic acids) in a DNA or RNA molecule that can potentially encode a peptide or a protein: said open reading frame begins with a START codon (the START codon generally encoding a methionine), followed by a series of codons (each codon encoding an amino acid), and ends with a STOP codon (the STOP codon not being translated).
  • START codon the START codon generally encoding a methionine
  • a series of codons each codon encoding an amino acid
  • STOP codon the STOP codon not being translated
  • the coding region of the mRNA therefore corresponds to the genetic sequence delimited by the START codon or initiation codon (most often encoding a methionine) at the 5' end and by the STOP codon at the 3' end.
  • the coding region of the mRNA therefore does not include the intronic sequences possibly present in the sequence of a gene or pre-messenger RNA (pre-mRNA), nor the 5'UTR and 3'UTR regions, because those -these are not translated and therefore do not encode part of the functional protein of the gene.
  • the sequence of an altPEP is determined by carrying out a translation of a fragment of the mRNA of the protein whose accumulation we want to modulate, said fragment being chosen from the (coding) sequence of nucleic acids naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
  • the same nucleic acid sequence naturally translated in said plant cell can give different altPEPs depending on the mRNA fragment chosen.
  • this same mRNA fragment can also give different altPEPs depending on the reading frame used to translate it, /.e. according to the grouping of the nucleotides of the sequence into consecutive triplets. Indeed and as previously mentioned, a translation can be carried out in two different reading frames (+2 and/or +3) thus potentially leading to two different altPEPs.
  • the “+1” reading frame corresponds to the reading frame determined by the initiation codon of the protein, /.e. the START codon of the open reading frame used naturally for mRNA translation.
  • the “+2” and “+3” reading frames correspond to reading frames which are not or only rarely used naturally for mRNA translation.
  • the reading frame “+2” corresponds to the reading frame shifted by one nucleotide in 3' (or two nucleotides in 5') compared to the open reading frame used naturally for the translation of the mRNA and the protein it codes .
  • the “+3” reading frame corresponds to the reading frame shifted by two nucleotides at 3' (or one nucleotide at 5') relative to the open reading frame used naturally for the translation of the mRNA and the protein it encodes.
  • the altPEPs obtained have a sequence different from that of a fragment of the amino acid sequence of the protein naturally encoded by said mRNA.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and in which said fragment is chosen in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or of two nucleotides 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the method for preparing and determining an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in 3' (or of a nucleotide at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5 ') or two nucleotides 3' (or one nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said altPEP is a hydrophobic peptide or a hydrophilic peptide.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said altPEP is a hydrophobic peptide.
  • hydrophobic peptide is meant a peptide whose amino acid sequence comprises more than 50% hydrophobic amino acids.
  • more than 50% we mean that the amino acid sequence comprises more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80% of amino acids. hydrophobic.
  • more than 50% we also mean that the amino acid sequence comprises at least 51%, at least 56%, at least 61%, at least 66%, at least 71%, at least 76% or at least 81% hydrophobic amino acids.
  • hydrophobic amino acids is meant the amino acids chosen from: alanine (Ala / A), isoleucine (Ile / 1), leucine (Leu / L), methionine (Met / M), phenylalanine (Phe / F), tryptophan (Trp/W), tyrosine (Tyr/Y) and valine (Val/V).
  • the invention also relates to the method for preparing and determining an altPEP as described above, in which said altPEP is a hydrophilic peptide.
  • hydrophilic peptide is meant a peptide whose amino acid sequence comprises more than 50% hydrophilic amino acids.
  • more than 50% we mean that the amino acid sequence comprises more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80% of amino acids. hydrophilic.
  • more than 50% we also mean that the amino acid sequence comprises at least 51%, at least 56%, at least 61%, at least 66%, at least 71%, at least 76% or at least 81% hydrophilic amino acids.
  • hydrophilic amino acids we mean the amino acids chosen from: aspartic acid (Asp / D), glutamic acid (Glu / E), arginine (Arg / R), asparagine (Asn / N), glutamine (Gin / Q ), histidine (His I H), lysine (Lys/K), serine (Ser/S) and threonine (Thr/T).
  • an altPEP can be produced by any type of means accessible to those skilled in the art.
  • an altPEP can be produced both by synthesis and by recombinant expression in homologous or heterologous systems.
  • the altPEP thus produced can then be introduced into a cell to modulate the accumulation of a target protein.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which, in step d., the production of said peptide is carried out by peptide synthesis or by recombinant expression.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which, in step d., the production of said peptide is carried out using an acid nucleic acid encoding said peptide introduced into a cell.
  • the invention relates to the process for preparing and determining an altPEP as described above, in which, in step e., the production of said peptide is carried out using an acid nucleic acid encoding said peptide introduced into said plant cell or into said plant.
  • the invention relates to a method for preparing and determining an altPEP as described above, in which, in step e., said peptide is brought into contact with said plant cell or in said plant .
  • the invention relates to the method for preparing and determining an altPEP as described above, in which, in step e., said peptide is present in said plant cell or in said plant following the expression of a nucleic acid encoding said peptide in said plant cell or in said plant.
  • another embodiment of the invention relates to the method of preparing and determining an altPEP as described above, in which, in step e., the presence of said peptide in said plant cell or in said plant results:
  • An altPEP can be used to modulate the accumulation of a protein present naturally (/e. endogenous) or not (/e. exogenous) in said plant cell or in said plant.
  • a “protein naturally present in a plant cell or in a plant” corresponds to an endogenous protein encoded by a gene present on the genome of the plant cell or the plant without the need for direct or indirect intervention by 'a human.
  • a “protein which is not naturally present in a plant cell or in a plant” corresponds to an exogenous protein encoded by a nucleic acid sequence present on the genome of the plant cell or plant which required the intervention of a human being and the use of means known to those skilled in the art.
  • a nucleic acid sequence may come from the same plant species or from a different plant species.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is of endogenous origin in said plant cells or said plants used in step e.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is of exogenous origin in said plant cells or said plants used in step e., said plant cells or said plants used in step e. then comprising a nucleic acid sequence allowing the expression of said protein.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blot, measurement of enzyme activity, mass spectrometry and translational fusion.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
  • step e The inventors have surprisingly noted that the use of altPEPs makes it possible to modify the phenotypes of a plant visible on a macroscopic scale. It is therefore entirely possible to use the latter to affirm (or refute) that the peptide determined in steps a., b. and c., and optionally produced in step d. is an altPEP (or not). This is also what the so-called phenotypic comparison alternative implemented in step e allows.
  • the term "plant” refers generally: to a set of plant cells organized in all or part of a plant whatever its stage of development (including the plant in the form of seed or young grows); to one or more organs of the plant (such as leaves, roots, stem, flowers); to one or more cells of the plant; or even a mass of plant cells ⁇ eg a callus).
  • the term "phenotype" designates in a non-limiting manner the characters visible on a macroscopic scale such as the number of lateral roots, the number of leaves, the size of the stem, the duration of flowering and the resistance to stress.
  • the invention therefore relates to the method for preparing and determining an altPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • a protein is “involved in a phenotype” if a modification of the accumulation thereof is associated with a modification of said phenotype.
  • a protein is involved in a phenotype if it intervenes in the character(s) corresponding to said phenotype.
  • an object of the invention is the process for preparing and determining an altPEP as described above, in which the phenotype observed in step e. is chosen from: the size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • the invention relates to the method for preparing and determining an altPEP as described above, said altPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, and in which said plant cell (/e.
  • Alopecurus myosuroides Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said plant cells or said plants used in step e. belong to: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora , Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) is a cell of an algae.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said plant cells or said plants used in step e belong to an alga.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene chosen from: Aae15 (Acyl-activating enzyme 15), Aae16 (AMP -dependent synthetase and ligase family protein), Abcg11 (White-brown complex-like protein), Abdcg34 (ABC transporter G family member 34), Acc1 (Acetyl-CoA Carboxylase), Agb1 (GTP binding protein beta 1), Als (Acetolactate synthase (chloroplastic)), Anac076 (NAC domain-containing protein 76), Apg9 (Autophagy 9), Arlbl (GTP-binding protein 1), Arr1 (Two-component response regulator ARR1), Arr5 (Two-component response regulator ARR5) , Arr6 (Two-component response regulator ARR6), At59 (Pedate lyase family protein), Bak1 (Brass) (A
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene chosen from: Cpk3, Dell and Nsp1.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by the Cpk3 gene.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by the Dell gene.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by the Nsp1 gene.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity. , preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NO: 1 (ORF of the Aae15 protein, A. thaliana), SEQ ID NO: 2 (ORF of the Aae16 protein, A. thaliana), SEQ ID NO: 3 (ORF of the abcg11 protein, A. thaliana), SEQ ID NO: 4 (ORF of the Abdcg34 protein, A. thaliana), SEQ ID NO: 5 (ORF of the Acc1 protein, A .
  • SEQ ID NO: 14 ORF of the protein At59, A. thaliana), SEQ ID NO: 15 (ORF of the Bak1 protein, A. thaliana), SEQ ID NO: 16 (ORF of the Bccpl protein, A. thaliana), SEQ ID NO: 17 (ORF of the Bccp2 protein, A. thaliana), SEQ ID NO: 18 (ORF of the Bri1 protein, A. thaliana), SEQ ID NO: 19 (ORF of the Bzo2h3 protein, A. thaliana), SEQ ID NO: 20 (ORF of the Cesa6 protein, A. thaliana), SEQ ID NO: 21 (ORF of the Cipk3 protein, A.
  • SEQ ID NO: 22 ORF of the Cks1 protein, A thaliana
  • SEQ ID NO: 23 ORF of the Cobl8 protein, A thaliana
  • SEQ ID NO: 24 ORF of the Coi1 protein, A. thaliana
  • SEQ ID NO: 25 ORF of the Coil protein, A. thaliana
  • SEQ ID NO: 26 ORF of the Cpk3 protein, A. thaliana
  • SEQ ID NO: 27 ORF of the Cpk3 protein, A. hypochondriacus
  • SEQ ID NO: 28 ORF of the Cpk3 protein, B. distachyori
  • SEQ ID NO: 29 ORF of the Cpk3 protein, B.
  • SEQ ID NO: 30 ORF of the Cpk3 protein, G. max
  • SEQ ID NO: 31 ORF of the Cpk3 protein, G. max
  • SEQ ID NO: 32 ORF of the Cpk3 protein, G. max
  • SEQ ID NO: 33 ORF of the Cpk3 protein, G. max
  • SEQ ID NO: 34 ORF of the Cpk3 protein, O. sativa
  • SEQ ID NO: 35 ORF of the Cpk3 protein, O. sativa
  • SEQ ID NO: 36 ORF of the Cpk3 protein, S. lycopersicum
  • SEQ ID NO: 37 ORF of the Cpk3 protein, Z.
  • SEQ ID NO: 38 ORF of the Cpk3 protein, Z. mays
  • SEQ ID NO: 39 ORF of the Cpk3 protein, Z. mays
  • SEQ ID NO: 40 ORF of the Cpk3 protein, B. rapa
  • SEQ ID NO: 41 ORF of the Cpk3 protein, B. rapa
  • SEQ ID NO: 42 ORF of the Cpk3 protein, H. vulgare
  • SEQ ID NO: 43 ORF of the Cpk3 protein, H. vulgare
  • SEQ ID NO: 44 ORF of the Cpk3 protein, S. tuberosum
  • SEQ ID NO: 45 ORF of the Cpk3 protein, A.
  • SEQ ID NO: 46 ORF of the Cpk3 protein, M. truncatula
  • SEQ ID NO: 47 ORF of the Cpk3 protein, M. truncatula
  • SEQ ID NO: 48 ORF of the Cpk3 protein, T. aestivum
  • SEQ ID NO: 49 ORF of the Cpk3 protein, T. aestivum
  • SEQ ID NO: 50 ORF of the Cpk3 protein, T. aestivum
  • SEQ ID NO: 51 ORF of the Cpk3 protein, T. aestivum
  • SEQ ID NO: 52 ORF of the Cpk3 protein, L.
  • SEQ ID NO: 53 ORF of the Cpk3 protein, L. perenne
  • SEQ ID NO: 54 ORF of the Cpk3 protein, L . perenne
  • SEQ ID NO: 55 ORF of the Cpk3 protein, L. perenne
  • SEQ ID NO: 56 ORF of the Crk34 protein, A. thaliana
  • SEQ ID NO: 57 ORF of the Cyp705a18 protein, A. thaliana
  • SEQ ID NO: 58 ORF of the Cyp71b26 protein, A. thaliana
  • SEQ ID NO: 59 ORF of the Cyp78a8 protein, A. thaliana
  • SEQ ID NO: 60 ORF of the Cyp97b3 protein , A.
  • SEQ ID NO: 61 ORF of the Dell protein, A. thaliana
  • SEQ ID NO: 62 ORF of the Dell protein, A. thaliana
  • SEQ ID NO: 63 ORF of the protein Dell, A. hypochondriacus
  • SEQ ID NO: 64 ORF of the Dell protein, B. distachyon
  • SEQ ID NO: 65 ORF of the Dell protein, G. max
  • SEQ ID NO: 66 ORF of the Dell protein, G. max
  • SEQ ID NO: 67 ORF of the Dell protein, O. sativa
  • SEQ ID NO: 68 ORF of the Dell protein, S.
  • SEQ ID NO: 69 ORF of the Dell protein, Z. mays
  • SEQ ID NO: 70 ORF of the Dell protein, B. rapa
  • SEQ ID NO: 71 ORF of the Dell protein, H. vulgare
  • SEQ ID NO: 72 ORF of the Dell protein, S. tuberosum
  • SEQ ID NO: 73 ORF of the Dell protein, M. truncatula
  • SEQ ID NO: 74 ORF of the Dell protein, T. aestivum
  • SEQ ID NO: 75 ORF of the Dell protein, T. aestivum
  • SEQ ID NO: 76 ORF of the Dell protein, T.
  • SEQ ID NO: 77 ORF of the Dell protein, T. aestivum
  • SEQ ID NO: 78 ORF of the Dell protein, L. perenne
  • SEQ ID NO: 79 ORF of the Dell protein, L. perenne
  • SEQ ID NO: 80 ORF of the Dur3 protein, A thaliana
  • SEQ ID NO: 81 ORF of the Ein2 protein, A. thaliana
  • SEQ ID NO: 82 ORF of the Emb175 protein, A. thaliana
  • SEQ ID NO: 83 ORF of the Emb2726 protein, A. thaliana
  • SEQ ID NO: 84 ORF of the Emb9 protein, A.
  • SEQ ID NO: 85 ORF of the Epsps protein, A. thaliana
  • SEQ ID NO: 86 ORF of the Fnr1 protein, A. thaliana
  • SEQ ID NO: 87 ORF of the Fve protein, A. thaliana
  • SEQ ID NO: 88 ORF of the Ga2ox7 protein, A. thaliana
  • SEQ ID NO: 89 ORF of the Gape protein, N. benthamiana
  • SEQ ID NO: 90 ORF of the Gcn2 protein, A. thaliana
  • SEQ ID NO: 91 ORF of the Gdi2 protein, A. thaliana
  • SEQ ID NO: 92 ORF of the Gln2 protein, A.
  • SEQ ID NO: 109 ORF of the Nsp1 protein, M. truncatula
  • SEQ ID NO: 110 ORF of the Nsp1 protein, A. thaliana
  • SEQ ID NO: 111 ORF of the Nsp1 protein, B. distachyon
  • SEQ ID NO: 112 ORF of the Nsp1 protein, G. max
  • SEQ ID NO: 113 ORF of the Nsp1 protein, G. max
  • SEQ ID NO: 114 ORF of the Nsp1 protein, O. sativa
  • SEQ ID NO: 115 ORF of the Nsp1 protein, S.
  • SEQ ID NO: 116 ORF of the Nsp1 protein, S. lycopersicum
  • SEQ ID NO: 117 ORF of the Nsp1 protein, Z. mays
  • SEQ ID NO: 118 ORF of the Nsp1 protein, Z. mays
  • SEQ ID NO: 119 ORF of the Nsp1 protein, Z. mays
  • SEQ ID NO: 120 ORF of the Nsp1 protein, Z. mays
  • SEQ ID NO: 121 ORF of the Nsp1 protein, B. rapa
  • SEQ ID NO: 122 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 123 ORF of the Nsp1 protein, H.
  • SEQ ID NO: 124 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 125 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 126 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 127 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 128 ORF of the Nsp1 protein, H. vulgare
  • SEQ ID NO: 129 ORF of the Nsp1 protein, S. tuberosum
  • SEQ ID NO: 130 ORF of the Nsp1 protein, S. tuberosum
  • SEQ ID NO: 131 ORF of the Nsp1 protein, T.
  • SEQ ID NO: 132 ORF of the Nsp1 protein, T. aestivum
  • SEQ ID NO: 133 ORF of the Nsp1 protein, L. perenne
  • SEQ ID NO: 134 ORF of the Nsp1 protein, L. perenne
  • SEQ ID NO: 135 ORF of the Pds protein, A. thaliana
  • SEQ ID NO: 136 ORF of the Pen3 protein, A. thaliana
  • SEQ ID NO: 137 ORF of the Phyb protein, A. thaliana
  • SEQ ID NO: 138 ORF of the Pif3 protein, A.
  • SEQ ID NO: 139 ORF of the Pizza protein, A thaliana
  • SEQ ID NO: 140 ORF of the Ppoxl protein, A thaliana
  • SEQ ID NO: 141 ORF of the Ppox2 protein, A. thaliana
  • SEQ ID NO: 142 ORF of the Prp39 protein, A. thaliana
  • SEQ ID NO: 143 ORF of the PsbA protein, A. thaliana
  • SEQ ID NO: 144 ORF of the Pskrl protein, A. thaliana
  • SEQ ID NO: 145 ORF of the Rd21 protein, A. thaliana
  • SEQ ID NO: 146 ORF of the Ringl protein, A.
  • SEQ ID NO: 147 ORF of the Rosi protein, A. thaliana
  • SEQ ID NO: 148 ORF of the Rpt4a protein, A. thaliana
  • SEQ ID NO: 149 ORF of the Sfr6 protein, A. thaliana
  • SEQ ID NO: 150 ORF of the Shr protein, A. thaliana
  • SEQ ID NO: 151 ORF of the Shy2 protein, A. thaliana
  • SEQ ID NO: 152 ORF of the Ski protein, M. truncatula
  • SEQ ID NO: 153 ORF of the Sps1 protein, A. thaliana
  • SEQ ID NO: 154 ORF of the Spt protein, A.
  • SEQ ID NO: 155 ORF of the Stn8 protein, A. thaliana
  • SEQ ID NO: 156 ORF of the Tap46 protein, A. thaliana
  • SEQ ID NO: 157 ORF of the Topp6 protein, A. thaliana
  • SEQ ID NO: 158 ORF of the TubB6 protein, A. thaliana
  • SEQ ID NO: 159 ORF of the TubB8 protein, A. thaliana
  • SEQ ID NO: 160 ORF of the llbala protein, A. thaliana
  • SEQ ID NO: 161 ORF of the Vim3 protein, A. thaliana
  • SEQ ID NO: 381 ORF of the Sgr1 protein, A.
  • SEQ ID NO: 382 ORF of the Abi5 protein, A. thaliana
  • SEQ ID NO: 383 ORF of the Hsp101 protein, A. thaliana
  • SEQ ID NO: 384 ORF of the Rh10 protein, M. truncatula
  • SEQ ID NO: 385 ORF of the Wus protein, A. thaliana
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity. , preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1 ).
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • percentage of identity between two nucleic acid (or amino acid) sequences, we mean a percentage of nucleotides (or amino acid residues) identical between the two sequences to be compared, obtained after the best alignment. . This percentage is purely statistical and the differences between the two sequences are randomly distributed over the entire length of the sequences.
  • the best alignment is the alignment for which the percentage of identity between the two sequences to be compared, as calculated below, is the highest.
  • Sequence comparisons between two nucleic acid (or amino acid) sequences are traditionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out per segment or per comparison window to identify and compare the regions local sequence similarity.
  • the optimal alignment of sequences for comparison can be carried out manually or by means of algorithms and software available to those skilled in the art, for example, the BLAST platform or the MatGat program (Campanella, Bitincka and Smalley , 2003).
  • the percentage identity between two sequences is determined by comparing these two optimally aligned sequences by comparison window in which the region of the sequence to be compared may include additions or deletions relative to the reference sequence for optimal alignment between these two sequences. Percent identity is calculated by determining the number of identical positions for which the nucleotide (or amino acid) is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100.
  • sequences presenting “at least 80% identity” with a reference sequence may in particular present at least 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% d identity with said reference sequence.
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs : 1 to 161 and 381 to 385.
  • the invention also relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a sequence of nucleic acids chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method for preparing and determining an altPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NO: 224 (AtDCL1alt18), SEQ ID NO: 225 (CrDCL1alt18), SEQ ID NO: 226 (EsDCL1alt18), SEQ ID NO: 227 (AIDCL1alt19), SEQ ID NO: 228 (CsDCL1alt19), SEQ ID NO: 229 (RsDCL1alt15), SEQ ID NO: 230 (BnDCL1alt14 ), SEQ ID NO: 231 (BoDCL1alt15), SEQ ID NO: 232 (BRDCL1alt17), SEQ ID NO: 233 (BsEIN2alt1), SEQ ID NO: 234 (CgEIN2alt1), SEQ ID NO: 235 (CrEIN2alt1), SEQ ID NO : 236 (EsEIN2alt1), SEQ ID NO: 237
  • the subject of the invention above is an altPEP as obtained by implementing the method as described above.
  • the invention also relates to an isolated altPEP, of 4 to 70 amino acids, in particular of 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a naturally translated fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to altPEP isolated as previously described, said fragment having a size of 3n nucleotides, n being included: from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
  • the invention relates to isolated altPEP as described above, said isolated altPEP comprising s, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 acids amines.
  • the invention relates to isolated altPEP as described above, said isolated altPEP comprising from 4 to 41 amino acids.
  • the invention relates to isolated altPEP as described above, said isolated altPEP comprising from 5 to 40 amino acids.
  • the invention relates to isolated altPEP as described above, said isolated altPEP comprising from 7 to 20 amino acids.
  • the invention also relates to isolated altPEP as described above, said isolated altPEP comprising from 8 to 15 amino acids.
  • the invention relates to isolated altPEP as described above, in which the size of said isolated altPEP is less than that of said protein.
  • the invention relates to altPEP isolated as described above, said fragment comprising: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention relates to altPEP isolated as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally occurring nucleic acid sequence. translated into said plant cell.
  • the invention relates to altPEP isolated as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to altPEP isolated as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5' ) relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the isolated altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3 ' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to isolated altPEP as described above, wherein said isolated altPEP is capable of increasing the accumulation of said protein in said plant cell.
  • the invention relates to isolated altPEP as described above, wherein said isolated altPEP is capable of reducing the accumulation of said protein in said plant cell.
  • the invention relates to isolated altPEP as described above, wherein said isolated altPEP is a synthetic peptide.
  • the invention relates to altPEP as described above, in which said isolated altPEP is a recombinant peptide.
  • the invention relates to isolated altPEP as described above, said isolated altPEP being a hydrophobic peptide or a hydrophilic peptide.
  • the invention relates to altPEP isolated as described above, in which said protein is naturally present in said plant cell.
  • the invention relates to altPEP isolated as described above, in which said protein is not naturally present in said plant cell.
  • the invention relates to isolated altPEP as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell.
  • the invention relates to isolated altPEP as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
  • the invention relates to the isolated altPEP as described above, in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) belongs to a plant species chosen from : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • the invention relates to isolated altPEP as described above, in which said plant cell is a cell of an alga.
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl , Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726 , Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gape, Gcn2, Gdi2, G/n2, Gs/3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2,
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene chosen from the genes: Cpk3, Dell and Nsp1.
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by the Cpk3 gene.
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by the Dell gene.
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by the Nsp1 gene.
  • the invention relates to isolated altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention relates to the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. % identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention also relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity.
  • the invention also relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention also relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention relates in particular to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a chosen among the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to altPEP isolated as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NOs: 224 to 366.
  • the invention relates to altPEP isolated as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and the number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • an altPEP may be fused or linked to one or more molecules facilitating entry of the altPEP into the cell.
  • these molecules we can notably cite peptides and palmitic acid.
  • penetrating peptides Numata, K., et al. Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Sci Rep 8 , 10966 (2018).
  • palmitic acid palmitic acid.
  • penetrating peptide hereinafter CPP
  • CPPs we mean small peptides that penetrate cellular lipid bilayers or destabilize cellular membranes.
  • CPPs can be classified into three groups: cationic, amphipathic and hydrophobic. Especially :
  • - cationic CPPs contain many positively charged amino acids, such as lysine (Lys) and arginine (Arg);
  • - Amphipathic CPPs are generally composed of an alternating sequence of polar and non-polar amino acids; And - Hydrophobic CPPs consist of nonpolar amino acids with relatively low net charges.
  • the invention relates to isolated altPEP as described above, said isolated altPEP being fused to a peptide facilitating its entry into the plant cell.
  • the invention relates to altPEP as described above, said altPEP being fused to a penetrating peptide.
  • the invention relates to isolated altPEP as described above, said isolated altPEP being fused at the N-terminus or at the C-terminus with said peptide facilitating its entry into the plant cell.
  • the invention relates to altPEP as described above, said altPEP being fused at the N-terminus or at the C-terminus with said penetrating peptide.
  • the invention relates to isolated altPEP as described above, said isolated altPEP being fused with: the TAT peptide (SEQ ID NO: 380); penetratin; a polyhistidine peptide (in particular a peptide of at least 4 histidine residues); or a polyarginine peptide (in particular a peptide of 4 arginine residues).
  • the invention relates to isolated altPEP as described above, said isolated altPEP being linked to one or more palmitic acid molecules.
  • the invention relates to isolated altPEP as described above, said isolated altPEP being linked at the N-terminus or at the C-terminus to one or more molecules of palmitic acid.
  • the quantity of altPEP necessary to modulate the accumulation of a protein can vary depending on whether or not altPEP is modified with one of the molecules facilitating its cellular penetration.
  • the invention above relates to a nucleic acid encoding an altPEP as described above.
  • the invention also relates to a nucleic acid of 3n nucleotides, which nucleic acid corresponds to a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA.
  • the invention relates to the nucleic acid as described above, said fragment comprising: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally occurring nucleic acid sequence. translated into said plant cell.
  • the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in the 3' (or by two nucleotides in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5' ) relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3 ' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the nucleic acid as described above, where n is comprised: from 4 to 70; from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
  • the above invention relates to a composition comprising an altPEP as described above as an active substance.
  • the above invention relates to a composition comprising an altPEP as active substance, said altPEP:
  • the invention relates to the composition as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the composition as described above, in which said altPEP is at a concentration comprised from 10' 9 M to 10' 3 M.
  • said composition of the invention does not exist in the natural state and this is all the more true since such a concentration of altPEP cannot exist within of a plant cell.
  • concentration comprised from 10' 9 M to 10' 3 M it is understood that the concentration of altPEP can be comprised from 10' 9 to 10' 4 M, from 10' 8 to 10' 4 M, from 10' 9 to 10' 5 M, from 10' 8 to 10' 5 M, as it can be comprised from 5 pM to 500 pM, from 30 pM to 70 pM, or even be 50 pM.
  • the invention relates to the composition as described above, in which said altPEP is at a concentration comprised from 10' 9 to 10' 4 M, from 10' 8 to 10' 4 M, from 10' 9 to 10' 5 M or from 10' 8 to 10' 5 M.
  • the invention relates to the composition as described above, in which said altPEP is at a concentration of from 5 pM to 500 pM or from 30 pM to 70 pM.
  • the invention relates to the composition as described above, in which said altPEP is at a concentration of 50 pM. In a non-limiting manner, this concentration can also be 10' 9 M, 10' 8 M, 10' 7 M, 10' 6 M, IO' 5 M or IO' 4 M.
  • the invention also relates to the composition comprising an altPEP as active substance, said altPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, of which the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA ; being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein; and being in particular at a concentration of 5 pM to 500 pM or from 30 pM to 70 pM, or being in particular at a concentration of 50 pM.
  • composition comprising an altPEP we mean that the composition of the invention comprises at least one altPEP. That is to say that a mixture of altPEPs is possible, said altPEPs being able to target the same protein or several proteins depending on the nucleic acid fragment from which they come.
  • the aforementioned concentrations concern either the mixture of altPEPs as such, or each of the altPEPs of said mixture, said altPEPs being able to be at the same concentration or being able to be at different concentrations among those cited above.
  • the invention relates to the composition as described above, said composition being a phytopharmaceutical composition, a herbicidal composition or a coating composition, in particular said coating composition further comprising at least one fixing agent .
  • the invention relates to the composition as described above, said composition being a phytopharmaceutical composition.
  • the invention relates to the composition as described above, said composition being a herbicidal composition.
  • the invention relates to the composition as described above, said composition being a coating composition.
  • the invention relates to the composition as described above, said composition being a coating composition further comprising at least one fixing agent.
  • the invention relates to the composition as described above, said composition further comprising at least one solvent.
  • said solvent is chosen from: acetone, acetonitrile, acetic acid, formic acid, dimethyl adipate, benzyl acetate, bi-butyl carbonate, dimethyl sulfoxide (DMSO), water, dimethyl glutarate, ammonium hydroxide, iso-butanol, iso-propanol, diethyl hexyl lactate, light aromatic solvent naphtha, heavy aromatic solvent naphtha, diethyl succinate and their mixtures (e.g.
  • altPEPs are determined in particular by their amino acid composition.
  • Hydrophilic altPEPs can be solubilized and packaged in aqueous solutions, such as water.
  • Hydrophobic altPEPs can be solubilized and packaged in solvents, such as organic solvents.
  • organic solvents are non-toxic solvents for plants in small quantities, that is to say they have no deleterious effect on the development of the plant.
  • the organic solvents may be those mentioned above and in particular chosen from acetonitrile and acetic acid.
  • altPEPs can also be solubilized and packaged in solvent mixtures, such as, for example, an organic solvent mixture [acetonitrile; acetic acid], a mixture [water; DMSO] in a volume:volume ratio of 99:1 to 1:99, a mixture [water; acetonitrile] in an inclusive volume:volume ratio of 99:1 at 1:99 or a mixture [water; ammonium hydroxide] in a volume:volume ratio of 99:1 to 99.9:0.1.
  • AltPEPs can also be solubilized in a solution comprising 50% acetonitrile, 10% acetic acid and 40% water (volume/volume/volume).
  • the invention relates to the composition as described above, said composition further comprising at least one diluent.
  • the invention relates to the composition as described above, said composition further comprising at least one adjuvant.
  • the invention relates to the composition as described above, said composition further comprising at least one fixing agent.
  • fixing agent is meant a chemical or natural agent which makes it possible to stick the composition of the invention to a plant seed so as to coat said plant seed.
  • a substance making it possible to apply and hold the active substance(s) on the grain.
  • fixing agents available, we find in particular carboxymethyl cellulose (CMC) and gum arabic.
  • a fixing agent may include organic solvents, water, dispersants, emulgators, surfactants, wetting agents and dyes.
  • the invention relates to the composition as described above, said composition further comprising at least one plant nutrient.
  • the invention relates to the composition as described above, said composition further comprising at least one fixing agent and at least one plant nutrient.
  • plant nutrient we mean an element assimilated by the plant to enable its development.
  • a plant nutrient can be chosen from: nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, manganese, iron, copper, boron, zinc, molybdenum and their mixtures.
  • a coated seed comprising a plant seed, said plant seed being coated with a coating composition as described above.
  • the coating can be carried out according to the methods conventionally used in the food industry and can be obtained by using a material capable of disintegrating in a solvent or in the earth, such as a binder or clay.
  • the coating can be used to confer particular properties to a seed in combination with an altPEP, such as improved growth or resistance to certain biotic or abiotic stresses.
  • the invention relates to the coated seed as described above, in which said plant seed has a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacu
  • the invention relates to coated seed as described above, said seed being treated by soaking in a composition containing an altPEP. During soaking, the seed is then totally or partially immersed in a composition containing an altPEP.
  • the invention relates to a use of an altPEP as a phytosanitary agent to modulate the accumulation of a protein in a plant cell, said altPEP having a size of 4 to 70 amino acids, in particular from 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the use of an altPEP as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention relates to the use of an altPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the sequence of nucleic acids naturally translated in said plant cell.
  • the invention relates to the use of an altPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the use of an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in 3' (or by one nucleotide in 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the use of an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the use of an altPEP as described above to increase the accumulation of said protein in the plant cell.
  • the presence of altPEP causes the quantity of said protein in the treated plant cell to be greater than that in an untreated plant cell.
  • the invention relates to the use of an altPEP as described above to reduce (inhibit) the accumulation of said protein in the plant cell.
  • the presence of altPEP causes the quantity of said protein in the treated plant cell to be lower than that in an untreated plant cell.
  • the invention relates to the use of an altPEP as described above, in which said altPEP is produced outside of said plant cell before being introduced into said plant cell. In one embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP is a synthetic peptide.
  • the invention relates to the use of an altPEP as described above, in which said altPEP is an isolated peptide.
  • the invention relates to the use of an altPEP as described above, in which said altPEP is a recombinant peptide.
  • the invention relates to the use of an altPEP as described above, in which said altPEP is a hydrophobic peptide or a hydrophilic peptide.
  • the invention relates to the use of an altPEP as described above, in which said altPEP is introduced into said plant cell in the form of a nucleic acid encoding said altPEP.
  • the invention relates to the use of an altPEP as described above, in which said altPEP is introduced into said plant cell in the form of a nucleic acid encoding said altPEP and comprising the means of expressing it.
  • the invention relates to the use of an altPEP as described above, in which said protein is naturally present in said plant cell.
  • the invention relates to the use of an altPEP as described above, in which said protein is not naturally present in said plant cell.
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell.
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
  • the invention relates to the use of an altPEP as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blotting, measurement of enzymatic activity, mass spectrometry and translational fusion.
  • the invention relates to the use of an altPEP as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
  • the invention relates to the use of an altPEP as described above, in which said altPEP has a size comprised from 4 to 41 amino acids, from 5 to 40 amino acids, from 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids.
  • said altPEP has a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
  • the invention relates to the use of an altPEP as described above, in which said plant cell belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochon
  • the invention relates to the use of an altPEP as described above, in which said plant cell is a cell of an alga.
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lr
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by an ORF comprising a sequence of nucleic acids having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention also relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the use of an altPEP as described above, in which said altPEP is chosen from the sequences: SEQ ID NOs: 224 to 366.
  • the invention relates to the use of an altPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume , the mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • plant phenotype chosen from: size, shape, surface area, volume , the mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral
  • the invention relates to the use of an altPEP as described above, to modulate the accumulation of a recombinant protein whose nucleic acid sequence which encodes it corresponds to the fusion of sequences d nucleic acids of two distinct genes.
  • the coding sequence of at least one of the two genes is that of a reporter gene, for example a gene encoding a fluorescent protein (such as GFP) or a protein allowing resistance of the plant to a compound.
  • a reporter gene for example a gene encoding a fluorescent protein (such as GFP) or a protein allowing resistance of the plant to a compound.
  • the invention relates to the use of an altPEP as described above, to modulate the accumulation of a recombinant protein whose nucleic acid sequence which encodes it corresponds to the fusion: of a nucleic acid sequence deemed non-coding of a first gene; And of a coding nucleic acid sequence of a second gene, the sequence of said altPEP corresponding to the translation via the genetic code of a fragment of the nucleic acid sequence deemed non-coding of the first gene.
  • the above invention relates to a method of modulating the accumulation of a protein in a plant cell comprising a step of introducing: an altPEP; or a nucleic acid encoding said altPEP and the means of expressing it, in said plant cell, the introduction of said altPEP leading to a modulation of the quantity of said protein in said plant cell, said altPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the method as described above, in which said fragment comprising: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or two nucleotides in 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relation to the frame open for reading the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the method as described above, said method making it possible to: promote the development of a plant; or slow down or prevent the development of a plant.
  • the invention relates to the process as described above, said process making it possible to promote the development of a plant.
  • the invention relates to the process as described above, said process making it possible to slow down or prevent the development of a plant.
  • the invention relates to the method as described above for increasing the accumulation of said protein in the plant cell.
  • the presence of altPEP causes the quantity of said protein in the treated plant cell to be greater than that in an untreated plant cell.
  • the invention relates to the method as described above for reducing (inhibiting) the accumulation of said protein in the plant cell.
  • the presence of altPEP causes the quantity of said protein in the treated plant cell to be lower than that in an untreated plant cell.
  • the invention relates to the method as described above, in which said altPEP is produced outside of said plant cell before being introduced into said plant cell.
  • the invention relates to the method as described above, in which said altPEP is a synthetic peptide.
  • the invention relates to the method as described above, in which said altPEP is an isolated peptide.
  • the invention relates to the method as described above, in which said altPEP is a recombinant peptide. In one embodiment, the invention relates to the method as described above, in which said altPEP is a hydrophobic peptide or a hydrophilic peptide.
  • the invention relates to the method as described above, in which said altPEP is introduced into said plant cell in the form of a nucleic acid encoding said altPEP.
  • the invention relates to the method as described above, in which said altPEP is introduced into said plant cell in the form of a nucleic acid encoding said altPEP and comprising the means of expressing it.
  • the invention relates to the method as described above, in which said protein is naturally present in said plant cell.
  • the invention relates to the method as described above, in which said protein is not naturally present in said plant cell.
  • the invention relates to the method as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell.
  • the invention relates to the method as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
  • the invention relates to the method as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blotting, measurement of enzymatic activity , mass spectrometry and translational fusion.
  • the invention relates to the method as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
  • the invention relates to the method as described above, in which said altPEP has a size comprised from 4 to 41 amino acids, from 5 to 40 amino acids, from 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids.
  • said altPEP has a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
  • the invention relates to the method as described above, in which said plant cell or said plant belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata , Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii , Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochon
  • the invention relates to the method as described above, in which said plant cell is an algae cell.
  • the invention relates to the method as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Aribl, Arr1 , Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9 , Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, G/n2, Gs/3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Map
  • the invention relates to the method as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
  • the invention relates to the method as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385. In one embodiment, the invention also relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell).
  • the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
  • the invention relates to the method as described above, in which said altPEP is chosen from the sequences: SEQ ID NOs: 224 to 366.
  • the invention relates to the method as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
  • plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration
  • the invention relates to the method as described above, in which the introduction of said altPEP results in early bolting in said plant.
  • the invention relates to the method as described above, in which the introduction of said altPEP results in earlier flowering in said plant.
  • the invention relates to the method as described above, in which the introduction of said altPEP causes an increase in the size of the stem in said plant.
  • the invention relates to the method as described above, in which the introduction of said altPEP results in earlier growth of the stem in said plant.
  • the inventors have in fact unexpectedly observed that it is possible to directly apply an altPEP to the plant, e.g. via the use of the composition of the invention (see above) comprising an altPEP, to modulate the accumulation of a target protein in the plant, indicating that altPEP is taken up by the plant.
  • the invention relates to the method as described above, in which said altPEP is introduced into said plant: by watering, by spraying or by adding a fertilizer, a potting soil, a culture substrate or a support in contact with the plant, said altPEP being in particular administered to the plant in the form of a composition comprising from IO' 9 M to IO' 4 M of said altPEP; by watering, by soaking, by spraying or by adding a fertilizer, a potting soil, a growing substrate or a support in contact with the plant, said altPEP being in particular administered to a seed or a seed in the form of a composition comprising from 10'9 M to 10'4 M of said altPEP; Or by means of a nucleic acid encoding said altPEP and comprising the means of expressing said altPEP, said nucleic acid being artificially introduced into the plant.
  • the invention relates to the method as defined above, in which said altPEP is artificially introduced externally into the plant, preferably by watering, by spraying or by the addition of a fertilizer, potting soil, a growing substrate or an inert support.
  • the invention relates to the method as defined above, in which said altPEP is introduced by watering.
  • the invention relates to the method as defined above, in which said altPEP is introduced by spraying.
  • the invention relates to the process as defined above, in which said altPEP is introduced by the addition of a fertilizer.
  • the invention relates to the process as defined above, in which the plant is treated with a composition comprising from 10' 9 M to 10' 4 M of said altPEP, or notably comprising 10' 9 M, 10' 8 M, 10' 7 M, 10' 6 M, 10' 5 M or 10' 4 M of said altPEP.
  • the compositions have a concentration of 10'8 M to 10'5 M for application by watering or spraying on the plant.
  • more or less concentrated compositions can be considered to treat the plant with altPEP.
  • more concentrated compositions comprising from 10' 1 M to 10' 3 M, or notably comprising 10' 2 M of altPEP, can be used in the case where the altPEP artificially introduced externally is administered to the plant by spreading.
  • the above invention relates to a modified plant containing an altPEP, which “modified plant” corresponds to a plant into which an altPEP has been artificially introduced, in particular by watering, by spraying or via fertilizer.
  • the invention relates to the modified plant comprising an exogenously introduced altPEP, said altPEP having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the modified plant as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally translated nucleic acid sequence. in said plant cell.
  • the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in the 5') or two nucleotides in the 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the modified plant as described above, said plant belonging to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus,
  • the above invention relates to a transgenic plant comprising a nucleic acid encoding an altPEP and the means for expressing it, said altPEP having a size of from 4 to 70 amino acids, in particular from 4 to 41 acids.
  • amino whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of an encoded protein by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
  • the invention relates to the transgenic plant as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
  • the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally translated nucleic acid sequence. in said plant cell.
  • the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention also relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3' (or one nucleotide in 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
  • the invention relates to the transgenic plant as defined above, in which the sequence encoding said altPEP is shorter than the sequence of the mRNA encoding said protein.
  • the invention relates to the transgenic plant as described above, said plant belonging to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp.
  • a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus
  • the invention relates to the transgenic plant as described above, in which the expression of said altPEP is placed under the control of a strong promoter, preferably a strong constitutive promoter such as the 35S promoter.
  • Figure 1 is a schematic representation illustrating the first steps of the process for preparing and determining a cPEP. In particular, the steps making it possible to determine within an mRNA of a protein one of the nucleic acid sequences from which the peptide to be tested (/e. the potential cPEP) is identified are illustrated.
  • sequences: SEQ ID NOs: 375 to 379 are provided as an example only.
  • cPEPs require sequence complementarity with their target for their activity
  • NSP1 AcPEP version of NSP1 in which the cPEP sequence has been deleted
  • NSP1 AcPEP- cPEPartificiel version of NSP1 in which the cPEP sequence was replaced by an artificial sequence
  • control control
  • cPEPnspl or cPEPartificiel an empty vector (control) or cPEPnspl or cPEPartificiel.
  • f,g Lateral root formation of seedlings of M. truncatula WT, nsp1 or overexpressing NSP1, in response to cPEPnspl or Scrambled cPEPnspl.
  • h,i Quantification of nodules on M. truncatula roots treated with an irrelevant peptide or cPEPskl.
  • cPEPs can modulate the development of A. thaliana and its response to stress
  • cPEPs can modulate the development of A. thaliana and its response to stress (a) Quantification of CPK3 expression, using CPK3 antibodies, in peptide-treated A. thaliana plants, (b-c) Test infection of peptide-treated ⁇ thaliana leaves inoculated with B. cinerea spores.
  • cPEPs are useful tools in agronomy
  • the plants of Medicago truncatula Gaertn cv. Jemalong genotype A17 were cultured on Long Aston medium as described in Delaux PM et al. (New Phytol. 2013 Jul;199(1):59-65).
  • Arabidopsis thaliana Col-0 plants were grown in soil until the age of 4 weeks in a growth chamber (22/20°C, 16 h/8 h L/D, RH 80%, ⁇ 75 pmol. m'2s'1 ) .
  • Barbarea vulgaris seeds were stratified for 24 h at 4°C before being grown in a pot in a growth chamber.
  • the peptides with sequences SEQ ID NOs: 162 to 223 and 386 to 418 were synthesized by Smart Biosciences and dissolved at a concentration of 2 to 10 mM in water, aliquoted and stored at - 80°C.
  • Plasmids were obtained using the Golden Gate cloning strategy in modified pCAMBIA220.
  • the Mt-NSP1 (Medtr8g020840):GUS translational fusion was performed using 3183 bp of the NSP1 promoter and 3180 bp of the post CDS section of NSP1. Expressions in N. benthamiana leaves were carried out using the 35S promoter.
  • the quantification of mRNAs was carried out by qRT-PCR using the appropriate pairs of primers chosen from the sequences: SEQ ID NOs: 367 to 374. Expression levels for the controls were set at 100.
  • the primers used for the tests on A. euteiches are described in Camborde et al. (New Phytol. 233:2232-2248. 2022).
  • Wild-type or mutant A thaliana leaves were sprayed daily with 100 pM peptide or 100 pM of the corresponding scramble version for 3 days.
  • Six hours after the last treatment five mature leaves per plant were inoculated with a 5 ⁇ L droplet of 2.5x10 5 spores/mL of Botrytis cinerea strain B05.10 diluted in 100 ⁇ M peptide or its corresponding scramble version .
  • plants were maintained in 100% relative humidity. Then, a 2 pL droplet of 100 pM peptide or 100 pM of the corresponding scramble version was deposited daily on B. cinerea-infected leaves for 3 days (until symptoms appeared).
  • Leaves were collected from the plants to determine lesion areas using the ImageJ program.
  • the protocol was the same except that the inoculation was carried out with 5,000 spores of B. cinerea, without peptide.
  • Peptides were added 1 h after inoculation (1 pL of 500 pM) and each day for two days with 5 pL of 100 pM peptide.
  • For infection of M. truncatula with A. Euteiches plants were grown on agar medium and treated with 10 ⁇ L of 100 ⁇ M peptides, 24 h before, and 24 h and 72 h after infection with 10 pL (1000 spores) of A. Euteiches. Plants were harvested 7 days after inoculation for RNA extraction.
  • N. benthamiana plants were treated by leaf spray 24 h before harvest.
  • 100 ⁇ L of MS/2 liquid medium containing the peptides was added to each well.
  • 5 ⁇ L of luciferin was added and luciferase activity was read 30 minutes later using a spectrophotometer. All other tests were carried out by spraying or watering the plants.
  • rosettes were sprayed daily with 100 pM peptide or its control for 5 days. Leaves were harvested 6 hours after the last treatment for Western blot analyses.
  • Plants were treated with 100 pM peptide for 24 h.
  • 3-day-old seedlings were treated for three days with 100 pM peptide before placing the plants at 45°C for 45 min. Plants were placed in a growth chamber to recover and treated 24 h after heat shock with 100 pM peptide.
  • 1-week-old seedlings were treated for 48 h, 24 h, and 30 min before being placed at 45°C for 24 h.
  • seedlings were treated 3 times per week for two weeks with 500 ⁇ L of 100 ⁇ M peptides.
  • the T nT® SP6 High Yield Wheat Germ Protein Expression System (Promega) was used according to the manufacturer's instructions.
  • the LUC sequence was amplified using GoTaq® polymerase (Promega), cPEPs or their corresponding Scramble versions were added just before the start of the reaction. The reaction was stopped after 60 min, after which LUC activity was measured with a plate spectrophotometer (Perkin-Elmer Victor Nivo).
  • Arabidopsis thaliana plants comprising the LUC construct were treated by infiltrating the leaves with a solution of peptide with or without cycloheximide (200 pg.rnL' 1 ) in MS/2 medium, 24 h before harvesting for the LUC test.
  • RNA co-immunoprecipitation was adapted from Merret et al. (Plant Physiol. 174:1216-1225. 2017). 400 mg of tissue powder was incubated in 3 mL of lysis buffer (200 mM Tris, pH 9.0, 110 mM potassium acetate, 0.5% Triton X-100, 0.1% Tween® 20.5 mM DTT, 1.5% protease inhibitor and 80 units ml-1 RNasin). The lysate was incubated on ice for 10 minutes, then centrifuged at 16,000 g for 10 minutes at 4°C.
  • lysis buffer 200 mM Tris, pH 9.0, 110 mM potassium acetate, 0.5% Triton X-100, 0.1% Tween® 20.5 mM DTT, 1.5% protease inhibitor and 80 units ml-1 RNasin.
  • the samples were prepared based on the protocols of Camborde et al (Nat Protoc. 12:1933-1950. 2017). Briefly, Agrobacterium tumefaciens strain GV3101 pmp90 carrying 35S-NSP1 or 35S-NSP1AORF plasmids were used to infiltrate N. benthamiana leaves. The agro-infiltrated discs of at least three different sheets were fixed after 48 hours by vacuum infiltration of a 4% (w/v) paraformaldehyde fixing solution, followed by a permeabilization step using of treatment with proteinase K. After washing, the staining of the nucleic acids was carried out by vacuum infiltration of a 5pM Sytox Orange solution (Invitrogen). Then, the disks were washed and mounted on TBS before FLIM measurements of the nuclei.
  • the 35s-NSP1 or 35s-NSP1AcPEP plasmids were transformed into A. tumefaciens GV3101 strain pmp90 and agro-infiltrated into N. benthamiana leaves. 40h later, a solution of 10pM cPEP-FAM was infiltrated into the same leaves and the plants were incubated for 3h. Next, leaf discs were fixed and processed as described in Camborde et al (Nat Protoc. 12:1933-1950. 2017), then washed and mounted on TBS before cytoplasmic FLIM measurements.
  • FLIM was carried out on Leica TCS SP8 SMD which consists of a LEICA DMi8 inverted microscope equipped with a TCSPC system from PicoQuant. Excitation of the FITC donor at 470 nm was carried out by a picosecond pulsed diode laser at a repetition rate of 40 MHz, through an oil immersion objective (63*, NA 1.4). The emitted light was detected by a Leica HyD detector in the emission range of 500–550 nm. Images were acquired with acquisition photons of up to 1500 per pixel. FLIM FRET analysis, FLIM data analysis
  • the decay curves were calculated per pixel and fitted (by maximum likelihood estimation of the Poisson Law) with a mono- or double-exponential decay model using the SymphoTime 64 software (PicoQuant, Germany).
  • the mono-exponential model function was applied for donor samples with only FITC present.
  • the double-exponential model function was used for samples containing FITC and Sytox. The experiments were repeated at least three times to obtain statistically valid data.
  • altPEPs control the translation of their coding gene
  • a first objective was to examine whether natural altPEPs could be detected in A. thaliana (Fig. 2a). To do this, a list of all potential altORFs of more than 10 amino acids in length, and located out of frame in the coding sequences of A. thaliana, was bioinformatically generated. Based on this list, we analyzed a recently published MS dataset (Müller JB et al. Nature. 582, 592-596 (2020)) and identified 112 novel altPEPs, showing that altPEPs are naturally represented in the proteome. in planta (SEQ ID NOs: 252-363). At the same time, 85 of the 112 corresponding reference proteins were identified in the same dataset, suggesting that altPEPs could in some cases be more expressed than their reference proteins.
  • altPEPs Two types of altPEPs were studied: one well conserved among the Brassicaceae, and corresponding to altORFI 8 of the DCL1 gene (Fig. 3c), and another, corresponding to the first altORF of the gene COI1, but not conserved among Brassicaceae.
  • Ae thaliana seedlings were treated with the peptide AtDCLI ait 18 (SEQ I D NO: 224) and root development analyzed, since dcl1 mutants have longer main roots (Park, W., et al. Curr. Biol. 12, 1484-1495 (2002)).
  • the first altPEPs of more than 10 amino acids of several well-known coding genes of A. thaliana were identified by bioinformatics. These are the three genes: AP2 and EIN2, for which the first potential altPEP was conserved among Brassicaceae (Fig. 3d, e), and CPK3, for which the first altPEP was not conserved. It was also tested whether altAUG 1 of the EIN2 gene is translated in vitro, using an in vitro transcription/translation system in wheat germ extracts. For this, the CDS of luciferase was fused to the 5'UTR of EIN2 up to refAUG or altAUGI, and the expression of luciferase was compared.
  • altAUGI was shown to be effective in promoting translation (Fig. 3f).
  • the translation efficiency is of the same order as the activity of refAUG, suggesting co-translation in similar amounts of the canonical EIN2 protein and altPEP.
  • the plants were then treated with the three altPEPs synthetic (AtAP2alt1; SEQ ID NO: 243, AtCPK3alt1 and AtEIN2alt1; SEQ ID NO: 241).
  • an increase in the reference protein was observed, without detecting any change in mRNA expression (Fig. 2g,h; Fig. 4b,c,d). This suggests that increased expression of reference proteins is likely a common property of altPEPs.
  • the conservation of altPEPs does not seem to be important for their activity.
  • RNA-IP RNA-IP method followed by PCR on plants treated with HA-tagged peptides
  • cPEPluc-HA HA-tagged peptides
  • SEQ ID NO: 387 a corresponding Scrambled peptide
  • Fig. 5a a corresponding Scrambled peptide
  • plants were treated with the synthetic cPEPluc and luciferase expression was analyzed. While qPCR analysis showed no effect on mRNA abundance (Fig. 5b), treatment with the peptide increased luciferase activity compared to treatments with water or non-specific luciferase peptides (Fig. 5c).
  • a 96-well microplate assay was developed, in which seeds were sown on half MS solid medium and 14-day-old seedlings were treated by adding 100 ⁇ L of liquid medium containing the peptides.
  • ten additional peptides of 10 amino acids targeting distinct sequences of the luciferase gene were developed (SEQ ID NOs: 388 to 397), in the three frames (ORF1 corresponds to the canonical luciferase protein), and all were able to increase luciferase activity (Fig. 5d).
  • 7 cPEPs of 5 to 60 amino acids SEQ ID NOs: 398 to 403 were designed and synthesized.
  • truncatula is homologous to AtEIN2, and ski mutants develop more nodules than wild-type plants (Penmetsa et al. Plant J. 55:580-595, 2008). Consistently, the application of a cPEPskl (SEQ ID NO: 411) led to a decrease in the amount of nodules (Fig. 6h, i). The RH 10 protein of M. truncatula was then targeted, which modulates the response of plants to a pathogenic oomycete, Aphanomyces Euteiches (Camborde et al. New Phytol. 233:2232-2248. 2022).
  • a cPEP targeting the ABI5 protein (SEQ ID NO: 406) could be identified, reducing the chlorophyll content, and a cPEP targeting the SGR1 protein (SEQ ID NO: 407), increasing this content (Fig. 7c ).
  • a cPEP targeting the HSP101 protein (SEQ ID NO: 404), involved in tolerance to heat stress (Queitsch et al, Plant Cell. 12:479-492, 2000), improved the viability of seedlings to shock. thermal (Fig. 7d).
  • cPEPs increase the efficiency of protein translation
  • cPEPs increase the amount of protein without disrupting mRNA levels, suggesting that cPEPs increase protein translation or stability.
  • A. thaliana plants expressing the LUC gene were treated with cPEPluc (SEQ ID NO: 213) and cycloheximide (CHX), a translation inhibitor. Luciferase activity assay showed that the effect of cPEPluc was inhibited in the presence of CHX, showing that cPEPs do not act on protein stability (Fig. 9a).
  • the LUC gene was expressed with or without cPEPluc in the wheat germ in vitro transcription/translation system, where no protease activity occurs. This revealed that cPEPluc increased LUC activity in vitro, strongly supporting the idea that cPEPs increase protein translation efficiency (Fig. 9b).
  • cPEPs could be the use in agronomy to improve crop yield.
  • they were tested on plants of agronomic interest, focusing on the same phenotypes as those that were studied on model plants.
  • the work first focused on plant defense and the tomato JAR1 protein was targeted.
  • treatment of tomato with cPEPjarl SEQ ID NO: 413) improved the plant's resistance to B. cinerea (Fig. 10a, b).
  • the homolog of HSP101 in soybean was identified and a cPEP targeting this protein was designed (SEQ ID NO: 408).
  • treatment of soybean plants with this peptide increased their tolerance to heat stress (Fig.
  • cPEPs were designed to target the corresponding proteins (respectively SEQ ID NOs: 204, 176, 177 and 196). A mixture of these cPEPs was able to decrease plant growth (Fig. 10i, j).
  • cPEPs cPEPs technology
  • the mode of action of cPEPs based on the complementarity of their sequence with the targeted protein, will make it possible to more easily identify non-specific interactions by bioinformatics, which will make it possible to target a single species, a family. or all plants.
  • peptides are short polymers of amino acids, they are likely to be quickly degraded by the soil microbiota, unlike polluting chemicals. Furthermore, the penetration of peptides into animal cells seems difficult without the presence of peptides penetrating into cells, which suggests that cPEPs will have no biological activity in animals and humans, apart from possible toxicity. intrinsic.

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Abstract

The present invention relates to novel peptides (cPEPs & altPEPs), to a method for the preparation thereof, and to the use thereof for modulating the accumulation of specific proteins.

Description

DESCRIPTION DESCRIPTION
TITRE : NOUVEAUX PEPTIDES ET LEUR UTILISATION POUR MODULER L’ACCUMULATION D’UNE PROTEINE TITLE: NEW PEPTIDES AND THEIR USE TO MODULATE THE ACCUMULATION OF A PROTEIN
DOMAINE DE L’INVENTION FIELD OF INVENTION
La présente invention concerne de nouveaux peptides (cPEPs & altPEPs), leur procédé de préparation et leur utilisation pour moduler l’accumulation de protéines spécifiques. The present invention relates to new peptides (cPEPs & altPEPs), their preparation method and their use to modulate the accumulation of specific proteins.
ART ANTERIEUR PRIOR ART
De manière générale, les peptides sont de courtes séquences de 2 à environ 100 acides aminés. Il s’agit souvent de molécules très actives, comme par exemple des hormones ou des composés de venins. Generally speaking, peptides are short sequences of 2 to approximately 100 amino acids. These are often very active molecules, such as hormones or venom compounds.
Chez les plantes, les peptides remplissent de nombreuses fonctions biologiques, telles que le développement ou les mécanismes de défense. Dans la mesure où la détection des peptides est relativement difficile, seul un nombre limité de peptides a été identifié, sous-estimant probablement la quantité et le rôle des peptides chez ces organismes. In plants, peptides perform many biological functions, such as development or defense mechanisms. Because peptide detection is relatively difficult, only a limited number of peptides have been identified, likely underestimating the quantity and role of peptides in these organisms.
La plupart des peptides caractérisés chez les plantes résulte vraisemblablement de la dégradation de protéines fonctionnelles. Toutefois, il a été montré que les transcrits primaires de microARNs (miRs) des plantes contiennent en réalité de petits cadres ouverts de lecture (miORFs) codant des peptides régulateurs, appelés miPEPs (Lauressergues D ét al. Primary transcripts of microRNAs encode regulatory peptides. Nature. 2015 Apr 2 ;520(7545):90-3.). Les miPEPs sont produits au même endroit que les miRs d’où ils proviennent et permettent d’améliorer la transcription des pri-miRs correspondants. L’activité d’un miPEP est très spécifique du mi R correspondant, permettant de sur-réguler le mi R choisi sans affecter l’expression des autres miRs. L’utilisation de miPEPs peut permettre de moduler l’expression d’un gène, si celui-ci est régulé par un miR, lui-même régulé par un miPEP (WO 2015/063431). Most of the peptides characterized in plants likely result from the degradation of functional proteins. However, it has been shown that primary transcripts of plant microRNAs (miRs) actually contain small open reading frames (miORFs) encoding regulatory peptides, called miPEPs (Lauressergues D et al. Primary transcripts of microRNAs encode regulatory peptides. Nature. 2015 Apr 2;520(7545):90-3.). The miPEPs are produced in the same place as the miRs from which they come and help improve the transcription of the corresponding pri-miRs. The activity of a miPEP is very specific to the corresponding mi R, making it possible to upregulate the chosen mi R without affecting the expression of other miRs. The use of miPEPs can make it possible to modulate the expression of a gene, if it is regulated by a miR, itself regulated by a miPEP (WO 2015/063431).
BREF APERÇU BRIEF OVERVIEW
Dans ce contexte, l’exposé ci-après offre un moyen universel, et facilement exploitable, pour moduler spécifiquement l’accumulation d’une protéine choisie chez une plante à l’aide soit d’un peptide non naturel (invention No. 1 ; cPEP), c’est-à-dire un peptide qui n’est pas naturellement produit par la plante, soit d’un peptide naturel (invention No. 2 ; altPEP), c’est- à-dire un peptide qui est naturellement produit par la plante. In this context, the presentation below offers a universal, and easily exploitable, means for specifically modulating the accumulation of a chosen protein in a plant using either a non-natural peptide (invention No. 1; cPEP), that is to say a peptide which is not naturally produced by the plant, or a natural peptide (invention No. 2; altPEP), that is to say a peptide which is naturally produced by the plant.
Quelle que soit l’invention considérée, l’un de leurs aspects est de proposer un procédé de préparation et de détermination d’un peptide « cPEP » ou « altPEP » capable de moduler l’accumulation (l’expression) d’une protéine spécifique dans une cellule végétale. Un second aspect de celles-ci est de proposer un procédé de modulation de l’accumulation d’une protéine chez une plante à l’aide d’un cPEP ou d’un altPEP. Un troisième aspect de celles-ci est de proposer l’utilisation d’un cPEP ou d’un altPEP pour moduler l’accumulation d’une protéine chez une plante. Un quatrième aspect de celles-ci est de proposer un procédé pour favoriser, ralentir ou empêcher le développement d’une plante. Un cinquième aspect de celles-ci est de proposer des peptides cPEP ou des peptides altPEP permettant de moduler l’accumulation d’une protéine chez une plante. D’autres aspects complémentaires de ces inventions concernent un acide nucléique codant un cPEP ou un altPEP, des compositions comprenant un cPEP ou un altPEP et des plantes modifiées ou transgéniques comprenant un cPEP ou un altPEP. Whatever the invention considered, one of their aspects is to propose a process for the preparation and determination of a “cPEP” or “altPEP” peptide capable of modulating the accumulation (expression) of a protein. specific in a plant cell. A second aspect of these is to propose a method of modulating the accumulation of a protein in a plant using a cPEP or an altPEP. A third aspect of these is to propose the use of a cPEP or an altPEP to modulate the accumulation of a protein in a plant. A fourth aspect of these is to propose a process to promote, slow down or prevent the development of a plant. A fifth aspect of these is to propose cPEP peptides or altPEP peptides making it possible to modulate the accumulation of a protein in a plant. Other complementary aspects of these inventions relate to a nucleic acid encoding a cPEP or an altPEP, compositions comprising a cPEP or an altPEP and modified or transgenic plants comprising a cPEP or an altPEP.
DESCRIPTION DETAILLEE DETAILED DESCRIPTION
Considérant la première invention (cPEP), un premier aspect de celle-ci concerne un procédé de préparation et de détermination d’un cPEP, ledit cPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés ; étant capable de moduler l’accumulation d’une protéine dans une cellule végétale ; et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, ledit procédé comprenant : a. une étape de détermination de la séquence d’acides nucléiques de l’ARN messager (ARNm) codant ladite protéine ; b. une étape de détermination au sein de cet ARNm de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine ; c. une étape de détermination au sein de cette séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale d’un fragment de celle-ci, ledit fragment ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ledit fragment ayant une taille inférieure à celle de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale ; d. une étape de production du peptide codé par ledit fragment ; et e. une étape de comparaison : Considering the first invention (cPEP), a first aspect thereof relates to a method for preparing and determining a cPEP, said cPEP: having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids ; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a. a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein; b. a step of determining within this mRNA the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein; vs. a step of determining within this nucleic acid sequence naturally translated in said plant cell a fragment thereof, said fragment having a size of 3n nucleotides, n being between 4 and 70, in particular n being between 4 and 41, and said fragment having a size smaller than that of the nucleic acid sequence naturally translated in said plant cell; d. a step of producing the peptide encoded by said fragment; summer. a comparison step:
- entre l’accumulation de ladite protéine dans une cellule végétale en présence dudit peptide et l’accumulation de ladite protéine dans une cellule végétale de même type en absence dudit peptide ; et/ou - between the accumulation of said protein in a plant cell in the presence of said peptide and the accumulation of said protein in a plant cell of the same type in the absence of said peptide; and or
- entre le phénotype d’une plante en présence dudit peptide et le phénotype d’une plante de même type en absence dudit peptide, dans laquelle : - between the phenotype of a plant in the presence of said peptide and the phenotype of a plant of the same type in the absence of said peptide, in which:
- une différence de la quantité de ladite protéine en présence dudit peptide par rapport à la quantité de ladite protéine en absence dudit peptide ; et/ou - a difference in the quantity of said protein in the presence of said peptide compared to the quantity of said protein in the absence of said peptide; and or
- une différence du phénotype en présence dudit peptide par rapport au phénotype en absence dudit peptide, indique que ledit peptide est un cPEP capable de moduler l’accumulation de ladite protéine dans une cellule végétale. - a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is a cPEP capable of modulating the accumulation of said protein in a plant cell.
La présente invention repose sur la constatation inattendue faite par les Inventeurs qu’il est possible de moduler spécifiquement l’accumulation d’une protéine à l’aide d’un peptide particulier non produit naturellement, dont la séquence correspond à la traduction (artificielle) d’un fragment de l’ARN messager (ARNm) codant ladite protéine, ledit fragment étant choisi sur la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine. The present invention is based on the unexpected observation made by the Inventors that it is possible to specifically modulate the accumulation of a protein using a particular peptide not produced naturally, the sequence of which corresponds to (artificial) translation of a fragment of messenger RNA (mRNA) encoding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
Dans l’invention, le terme « cPEP » (complementary peptide) désigne un peptide artificiel capable de moduler spécifiquement l’accumulation d’une protéine une fois introduit dans une cellule végétale. In the invention, the term “cPEP” (complementary peptide) designates an artificial peptide capable of specifically modulating the accumulation of a protein once introduced into a plant cell.
Selon l’invention, un cPEP n’est pas présent naturellement dans une cellule végétale. Cela signifie que la cellule végétale contient l’information du cPEP mais ne contient pas la séquence nucléique capable de permettre son expression. Seule la séquence peptidique du cPEP peut être déduite à partir de la séquence de l’ARNm codant ladite protéine dont on veut moduler l’accumulation. Un cPEP n’est présent dans une cellule végétale qu’une fois que celui-ci y a été introduit sous la forme d’un peptide ou sous la forme d’un acide nucléique codant ledit peptide. According to the invention, a cPEP is not naturally present in a plant cell. This means that the plant cell contains the cPEP information but does not contain the nucleic sequence capable of allowing its expression. Only the peptide sequence of cPEP can be deduced from the sequence of the mRNA coding said protein whose accumulation we want to modulate. A cPEP is only present in a plant cell once it has been introduced there in the form of a peptide or in the form of a nucleic acid encoding said peptide.
La spécificité du cPEP vis-à-vis d’une protéine cible (d’un gène cible) est déterminée par sa séquence d’acides aminés. En effet, la séquence d’un cPEP correspond à la traduction in silico (artificielle) d’un fragment de l’ARNm codant ladite protéine, ledit fragment étant choisi sur la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine. The specificity of cPEP towards a target protein (target gene) is determined by its amino acid sequence. Indeed, the sequence of a cPEP corresponds to the in silico (artificial) translation of a fragment of the mRNA coding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
La séquence peptidique d’un cPEP peut donc être déterminée à partir d’un fragment de l’ARNm codant ladite protéine en appliquant, dès le premier nucléotide dudit fragment, le code génétique attribuant à chaque triplet de nucléotides un acide aminé spécifique (AUC = Isoleucine, ACA = Thréonine, etc.). The peptide sequence of a cPEP can therefore be determined from a fragment of the mRNA encoding said protein by applying, from the first nucleotide of said fragment, the genetic code attributing to each triplet of nucleotides a specific amino acid (AUC = Isoleucine, ACA = Threonine, etc.).
Dans l’invention, le fragment de l’ARNm utilisé pour déterminer la séquence du cPEP peut être choisi dans les trois cadres de lecture existants sur la séquence de l’ARNm. En d’autres termes, un fragment peut être sélectionné dans les cadres de lecture +1 , +2 ou +3. Sur ce point, il est possible que les trois des cadres de lecture contiennent l’information d’un cPEP comme il est possible que seulement un des trois des cadres de lecture (le +1 , le +2 ou le +3) ou deux des trois des cadres de lecture (le +1 et le +2, le +1 et le +3, ou le +2 et le +3) contiennent l’information d’un cPEP. In the invention, the mRNA fragment used to determine the cPEP sequence can be chosen from the three existing reading frames on the mRNA sequence. In other words, a fragment can be selected in reading frames +1, +2 or +3. On this point, it is possible that the three reading frames contain the information of a cPEP as it is possible that only one of the three reading frames (the +1, the +2 or the +3) or two of the three reading frames (+1 and +2, +1 and +3, or +2 and +3) contain the information of a cPEP.
Dans l’invention, le terme « cadre de lecture » désigne le regroupement des nucléotides constituant une séquence d’acides nucléiques en triplets (ou codons) consécutifs, qui se succèdent sans interruption ni recouvrement. In the invention, the term “reading frame” designates the grouping of nucleotides constituting a nucleic acid sequence into consecutive triplets (or codons), which follow one another without interruption or overlap.
D’une manière générale, les cPEPs ont une taille comprise de 4 à 70 acides aminés, en particulier une taille comprise de 4 à 41 acides aminés, notamment une taille comprise de 5 àGenerally speaking, cPEPs have a size of 4 to 70 amino acids, in particular a size of 4 to 41 amino acids, in particular a size of 5 to 41 amino acids.
40 acides aminés, de 7 à 20 acides aminés ou plus particulièrement une taille comprise de 8 à 15 acides aminés. Par conséquent, la séquence d’un cPEP correspond à la traduction d’un fragment compris de 4 à 70 triplets de nucléotides, en particulier un fragment compris de 4 à40 amino acids, from 7 to 20 amino acids or more particularly a size of 8 to 15 amino acids. Consequently, the sequence of a cPEP corresponds to the translation of a fragment comprised of 4 to 70 nucleotide triplets, in particular a fragment comprised of 4 to
41 triplets de nucléotides, notamment d’un fragment compris de 5 à 40 triplets de nucléotides, de 7 à 20 triplets de nucléotides ou plus particulièrement d’un fragment compris de 8 à 15 triplets de nucléotides. 41 nucleotide triplets, in particular a fragment comprising 5 to 40 nucleotide triplets, 7 to 20 nucleotide triplets or more particularly a fragment comprising 8 to 15 nucleotide triplets.
En d’autres termes, la séquence d’un cPEP correspond à la traduction en acides aminés d’un fragment de « 3n » nucléotides de l’ARNm de la protéine cible, n étant compris de 4 à 70, en particulier compris de 4 à 41 , notamment compris de 5 à 40, de 7 à 20 ou plus particulièrement compris de 8 à 15. In other words, the sequence of a cPEP corresponds to the translation into amino acids of a fragment of “3n” nucleotides of the mRNA of the target protein, n being between 4 and 70, in particular included from 4 to 41, in particular included from 5 to 40, from 7 to 20 or more particularly included from 8 to 15.
Par exemple, si n est égal à 5, le cPEP a une taille de 5 acides aminés et correspond à la traduction d’un fragment de 15 (= 3 x 5) nucléotides. Par exemple, si n est égal à 40, le cPEP a une taille de 40 acides aminés et correspond à la traduction d’un fragment de 120 (= 3 x 40) nucléotides. Par exemple, si n est égal à 70, le cPEP a une taille de 70 acides aminés et correspond à la traduction d’un fragment de 210 (= 3 x 70) nucléotides. Etc. For example, if n is equal to 5, the cPEP has a size of 5 amino acids and corresponds to the translation of a fragment of 15 (= 3 x 5) nucleotides. For example, if n is equal to 40, the cPEP has a size of 40 amino acids and corresponds to the translation of a fragment of 120 (= 3 x 40) nucleotides. For example, if n is equal to 70, the cPEP has a size of 70 amino acids and corresponds to the translation of a fragment of 210 (= 3 x 70) nucleotides. Etc.
Les cPEPs ont une taille de 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 ou 70 acides aminés, et correspondent respectivement à la traduction de fragments de 12, 15, 18, 21 , 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 , 54, 57, 60, 63, 66, 69, 72, 75, 78, 81 , 84, 87, 90, 93, 96, 99, 102, 105, 108, 111 , 114, 117, 120, 123, 126, 129, 132, 135, 138, 141 , 144, 147, 150, 153, 156, 159, 162, 165, 168, 171 , 174, 177, 180, 183, 186, 189, 192, 195, 198, 201 , 204, 207 ou 210 nucléotides. The cPEPs have a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids, and correspond respectively to the translation of fragments 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84 , 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 138, 141, 144, 147, 150, 153, 156, 159 , 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207 or 210 nucleotides.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit fragment a une taille de 3n nucléotides, n étant compris : de 4 à 41 ; de 5 à 40 ; de 7 à 20 ; ou de 8 à 15. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said fragment has a size of 3n nucleotides, n being included: from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit peptide a une taille choisie parmi : 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26,In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said peptide has a size chosen from: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 ,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 et 70 acides aminés. 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 and 70 amino acids.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit cPEP a une taille inférieure à celle de ladite protéine (/.e. celle dont le cPEP module l’accumulation). In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said cPEP has a size smaller than that of said protein (/e. that of which the cPEP modulates the 'accumulation).
Comme indiqué plus haut, les cPEPs ont la capacité de moduler spécifiquement l’accumulation d’une protéine sans que l’accumulation de l’ARNm lui correspondant soit impacté. Autrement dit, l’ajout d’un cPEP dans une cellule végétale ne modifie pas la quantité d’ARNm permettant d’exprimer la protéine qu’il a la capacité de réguler, mais seulement la quantité de ladite protéine. As indicated above, cPEPs have the capacity to specifically modulate the accumulation of a protein without the accumulation of the mRNA corresponding to it being impacted. In other words, the addition of a cPEP in a plant cell does not modify the quantity of mRNA making it possible to express the protein that it has the capacity to regulate, but only the quantity of said protein.
Selon l’invention, le terme « protéine » désigne une séquence d’acides aminés dont l’information est codée par un gène présent sur le génome d’une cellule végétale. Par « gène », on désigne donc, notamment, la séquence d’acides nucléiques nécessaire à la synthèse de ladite protéine. Aussi, un gène comprend davantage que les nucléotides codant la séquence d’acides aminés de la protéine. Par exemple, un gène inclut les séquences d’ADN nécessaires à la synthèse d’un pré-messager (pré-ARNm), lequel est ensuite maturé par la machinerie cellulaire en un ARN messager (ARNm). Ce dernier peut alors être traduit, via les ribosomes, en une protéine. According to the invention, the term “protein” designates a sequence of amino acids whose information is encoded by a gene present on the genome of a plant cell. By “gene”, we therefore designate, in particular, the nucleic acid sequence necessary for the synthesis of said protein. Also, a gene includes more than the nucleotides encoding the amino acid sequence of the protein. For example, a gene includes the DNA sequences necessary for the synthesis of a pre-messenger (pre-mRNA), which is then processed by the cellular machinery into messenger RNA (mRNA). The latter can then be translated, via ribosomes, into a protein.
Au vu de ce qui précède, on comprend que l’ARN pré-messager (pré-ARNm) n’a pas subi d’épissage et est susceptible de contenir des introns, tandis que l’ARN messager (ARNm) mature peut avoir subi un épissage et ne contient que des exons. In view of the above, it is understood that pre-messenger RNA (pre-mRNA) has not undergone splicing and is likely to contain introns, while mature messenger RNA (mRNA) may have undergone a splice and contains only exons.
Pour préparer un cPEP capable de moduler l’accumulation d’une protéine, il convient de traduire un fragment de l’ARN m codant ladite protéine, ledit fragment étant choisi sur la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine, lequel ne comprend ni la région 5’-UTR, ni la région 3’-UTR de l’ARNm. To prepare a cPEP capable of modulating the accumulation of a protein, it is appropriate to translate a fragment of the mRNA encoding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein, which includes neither the 5'-UTR region nor the 3'-UTR region of the mRNA.
Dans l’invention, la « modulation » de l’accumulation d’une protéine désigne soit une augmentation de l’accumulation de ladite protéine (/.e. une augmentation de la quantité de protéine dans la cellule végétale), soit une diminution de l’accumulation de ladite protéine (/.e. une diminution de la quantité de protéine dans la cellule végétale). Autrement dit, un mode de réalisation de l’invention a pour objet le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite modulation de l’accumulation de ladite protéine induite par ledit cPEP est : une diminution de l’accumulation de ladite protéine ; ou une augmentation de l’accumulation de ladite protéine. In the invention, the “modulation” of the accumulation of a protein designates either an increase in the accumulation of said protein (/e. an increase in the quantity of protein in the plant cell), or a decrease in the accumulation of said protein (/e. a decrease in the quantity of protein in the plant cell). In other words, one embodiment of the invention relates to the method of preparing and determining a cPEP as described above, in which said modulation of the accumulation of said protein induced by said cPEP is: a reduction of the accumulation of said protein; or an increase in the accumulation of said protein.
L’augmentation et la diminution de l’accumulation de ladite protéine peuvent être mesurées et suivies à l’aide de méthodes bien connues de l’homme du métier, telles que le couplage de la protéine à un marqueur via l’utilisation de cassettes d’expression particulières, ou un Western blot. Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel à l’étape e., la quantité de protéine en présence dudit peptide est supérieure à la quantité de protéine en absence dudit peptide. En d’autres termes, en présence d’un cPEP favorisant l’augmentation de l’accumulation de la protéine, la traduction de l’ARNm correspondant est augmentée, ce qui conduit à une production plus importante de la protéine sans que soit modifiée la quantité dudit ARNm. The increase and decrease in the accumulation of said protein can be measured and monitored using methods well known to those skilled in the art, such as the coupling of the protein to a marker via the use of cassettes. particular expression, or a Western blot. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which in step e., the quantity of protein in the presence of said peptide is greater than the quantity of protein in the absence of said peptide. In other words, in the presence of a cPEP promoting increased accumulation of the protein, the translation of the corresponding mRNA is increased, which leads to greater production of the protein without modifying the quantity of said mRNA.
Dans un autre mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel à l’étape e., la quantité de protéine en présence dudit peptide est inférieure à la quantité de protéine en absence dudit peptide. En d’autres termes, en présence d’un cPEP favorisant la diminution de l’accumulation de la protéine, la traduction de l’ARNm correspondant est diminuée (inhibée), ce qui conduit à une production moins importante de la protéine sans que soit modifiée la quantité dudit ARNm. In another embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which in step e., the quantity of protein in the presence of said peptide is less than the quantity of protein in the absence of said peptide. In other words, in the presence of a cPEP promoting the reduction of the accumulation of the protein, the translation of the corresponding mRNA is reduced (inhibited), which leads to less production of the protein without either modified the quantity of said mRNA.
Dans l’invention, bien que le fragment de l’ARNm codant ledit cPEP soit situé au sein la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine, ledit fragment en tant que tel n’est pas traduit naturellement dans ladite cellule végétale. Ceci, quel que soit le cadre de lecture utilisé. L’existence d’un cPEP au sein de ladite cellule végétale est donc artificielle et a pour origine une action de l’Homme. Pour cela, il est possible soit d’introduire artificiellement ledit cPEP en tant que tel, soit d’introduire une cassette d’expression comprenant la séquence d’acides nucléiques codant ledit cPEP et les moyens de l’exprimer dans ladite cellule végétale. In the invention, although the fragment of mRNA encoding said cPEP is located within the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein, said fragment as such is not translated naturally in said plant cell. This, regardless of the reading frame used. The existence of a cPEP within said plant cell is therefore artificial and originates from human action. For this, it is possible either to artificially introduce said cPEP as such, or to introduce an expression cassette comprising the nucleic acid sequence encoding said cPEP and the means of expressing it in said plant cell.
Dans l’invention, la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale, laquelle comprend un fragment portant l’information d’un cPEP, est une région de l’ARNm qualifiée de « codante », c’est-à-dire qu’elle correspond à une région de l’ARNm qui code tout ou partie de la protéine fonctionnelle. Cette séquence correspond donc au cadre ouvert de lecture principal, lequel code la protéine dont on veut moduler l’accumulation. In the invention, the nucleic acid sequence naturally translated in said plant cell, which comprises a fragment carrying the information of a cPEP, is a region of the mRNA described as “coding”, i.e. say that it corresponds to a region of the mRNA which codes all or part of the functional protein. This sequence therefore corresponds to the main open reading frame, which codes the protein whose accumulation we want to modulate.
Dans l’invention, les termes « cadre ouvert de lecture » et « ORF » (open reading frame) sont équivalents, et peuvent être utilisés l’un pour l’autre. Ils correspondent à une séquence de nucléotides (acides nucléiques) dans une molécule d’ADN ou d’ARN pouvant potentiellement coder un peptide ou une protéine : ledit cadre ouvert de lecture débute par un codon START (le codon START codant généralement une méthionine), suivi d’une série de codons (chaque codon codant un acide aminé), et se termine par un codon STOP (le codon STOP n’étant pas traduit). La région codante de l’ARNm correspond donc à la séquence génétique délimitée par le codon START ou codon d’initiation (codant le plus souvent une méthionine) à l’extrémité 5’ et par le codon STOP à l’extrémité 3’. La région codante de l’ARNm ne comprend pas donc les séquences introniques éventuellement présentes dans la séquence d’un gène ou de l’ARN pré-messager (pré-ARNm), ni les régions 5’UTR et 3’UTR, car celles-ci ne sont pas traduites et ne codent donc pas une partie de la protéine fonctionnelle du gène. In the invention, the terms “open reading frame” and “ORF” (open reading frame) are equivalent, and can be used interchangeably. They correspond to a sequence of nucleotides (nucleic acids) in a DNA or RNA molecule that can potentially encode a peptide or a protein: said open reading frame begins with a START codon (the START codon generally encoding a methionine), followed by a series of codons (each codon encoding an amino acid), and ends with a STOP codon (the STOP codon not being translated). The coding region of the mRNA therefore corresponds to the genetic sequence delimited by the START codon or initiation codon (most often encoding a methionine) at the 5' end and by the STOP codon at the 3' end. The coding region of the mRNA therefore does not include the intronic sequences possibly present in the sequence of a gene or pre-messenger RNA (pre-mRNA), nor the 5'UTR and 3'UTR regions, because those -these are not translated and therefore do not encode part of the functional protein of the gene.
Dans l’invention, la séquence d’un cPEP est déterminée en effectuant une traduction (artificielle) d’un fragment de l’ARNm de la protéine dont on veut moduler l’accumulation, ledit fragment étant choisi sur la séquence (codante) d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine. Aussi, une même séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale peut donner des cPEPs différents selon le fragment de l’ARNm choisi. Par ailleurs, ce même fragment d’ARNm peut lui aussi donner des cPEPs différents selon le cadre de lecture utilisé pour le traduire (artificiellement), i.e. selon le regroupement des nucléotides de la séquence en triplets consécutifs. En effet et comme précédemment mentionné, une traduction peut être réalisée dans les trois cadres de lecture différents conduisant ainsi à potentiellement trois cPEPs différents. In the invention, the sequence of a cPEP is determined by carrying out an (artificial) translation of a fragment of the mRNA of the protein whose accumulation we want to modulate, said fragment being chosen from the (coding) sequence of nucleic acids naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein. Also, the same nucleic acid sequence naturally translated in said plant cell can give different cPEPs depending on the mRNA fragment chosen. Furthermore, this same mRNA fragment can also give different cPEPs depending on the reading frame used to translate it (artificially), i.e. depending on the grouping of the nucleotides of the sequence into consecutive triplets. Indeed and as previously mentioned, a translation can be carried out in the three different reading frames thus potentially leading to three different cPEPs.
Selon l’invention, le cadre de lecture “+1” correspond au cadre de lecture déterminé par le codon d’initiation de la protéine, i.e. le codon START du cadre ouvert de lecture utilisé naturellement pour la traduction de l’ARNm. En d’autres termes, dans le cas d’un fragment d’ARNm correspondant à une région codante et traduit selon le cadre de lecture +1 , le cPEP obtenu possède une séquence identique à celle d’un fragment de la séquence d’acides aminés de la protéine naturellement codée par ledit ARNm. According to the invention, the “+1” reading frame corresponds to the reading frame determined by the initiation codon of the protein, i.e. the START codon of the open reading frame used naturally for the translation of the mRNA. In other words, in the case of an mRNA fragment corresponding to a coding region and translated according to the +1 reading frame, the cPEP obtained has a sequence identical to that of a fragment of the acid sequence amino acids of the protein naturally encoded by said mRNA.
Les cadres de lecture “+2” et “+3” correspondent à des cadres de lecture qui ne sont pas (ou peu) utilisés naturellement pour la traduction de l’ARNm. Selon l’invention, le cadre de lecture “+2” correspond au cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture utilisé naturellement pour la traduction de l’ARNm et de la protéine qu’il code. Selon l’invention, le cadre de lecture “+3” correspond au cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture utilisé naturellement pour la traduction de l’ARNm et de la protéine qu’il code. The “+2” and “+3” reading frames correspond to reading frames which are not (or little) used naturally for mRNA translation. According to the invention, the “+2” reading frame corresponds to the reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to the open reading frame used naturally for the translation of the mRNA and the protein it encodes. According to the invention, the “+3” reading frame corresponds to the reading frame shifted by two nucleotides at 3' (or one nucleotide at 5') relative to the open reading frame used naturally for the translation of the mRNA and the protein it encodes.
Généralement, dans le cas d’un fragment d’ARNm correspondant à une région codante et traduit selon le cadre de lecture +2 ou +3, les cPEPs obtenus possèdent une séquence différente de celle d’un fragment de la séquence d’acides aminés de la protéine naturellement codée par ledit ARNm. Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et dans lequel ledit fragment est choisi : soit dans le même cadre de lecture que le cadre ouvert de lecture codant ladite protéine ; soit dans un cadre de lecture décalé d’un ou deux nucléotides par rapport au cadre ouvert de lecture codant ladite protéine. Generally, in the case of an mRNA fragment corresponding to a coding region and translated according to the +2 or +3 reading frame, the cPEPs obtained have a sequence different from that of a fragment of the amino acid sequence. of the protein naturally encoded by said mRNA. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and in which said fragment is chosen: either in the same reading frame as the open reading frame encoding said protein; either in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; et d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et dans lequel ledit fragment est choisi : soit dans le même cadre de lecture que le cadre ouvert de lecture codant ladite protéine ; soit dans un cadre de lecture décalé d’un ou deux nucléotides par rapport au cadre ouvert de lecture codant ladite protéine. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and in which said fragment is chosen: either in the same reading frame as the open reading frame encoding said protein; either in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein.
On comprend donc qu’un cPEP, selon les modes de réalisation ci-dessus, comprend soit un codon AUG (et pas de codon STOP), soit un codon STOP (et pas de codon AUG), soit aucun de ces deux éléments. En l’espèce, c’est l’homme du métier qui ajoute si besoin l’élément manquant ou ces éléments manquants pour permettre, de manière non limitative, soit de produire in vitro un cPEP par le biais, par exemple, d’un microorganisme puis de l’utiliser (dans une composition par exemple), soit d’en introduire la séquence et les moyens de l’exprimer via un vecteur dans une cellule de plante ou une plante. We therefore understand that a cPEP, according to the above embodiments, comprises either an AUG codon (and no STOP codon), or a STOP codon (and no AUG codon), or neither of these two elements. In this case, it is the person skilled in the art who adds, if necessary, the missing element or these missing elements to allow, in a non-limiting manner, either to produce a cPEP in vitro by means of, for example, a microorganism then use it (in a composition for example), or introduce the sequence and the means of expressing it via a vector in a plant cell or a plant.
Dans un mode de réalisation, l’invention concerne donc le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit fragment comprend un codon initiateur AUG codant une méthionine initiatrice et est dépourvu d’un codon STOP choisi parmi les codons : UAG, UGA et UAA. L’invention concerne également le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit fragment est dépourvu d’un codon initiateur AUG codant une méthionine initiatrice et comprend un codon STOP choisi parmi les codons : UAG, UGA et UAA. Au vu de ce qui précède, on comprend que dans un autre mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit ci-dessus, ledit cPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés ; étant capable de moduler l’accumulation d’une protéine dans une cellule végétale ; et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, ledit procédé comprenant : a. une étape de détermination de la séquence d’acides nucléiques de l’ARN messager (ARNm) codant ladite protéine ; b. une étape de détermination au sein de cet ARNm de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine ; c. une étape de détermination au sein de cette séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale d’un fragment non naturellement traduit de celle-ci, ledit fragment non naturellement traduit : In one embodiment, the invention therefore relates to the method for preparing and determining a cPEP as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA. The invention also relates to the method for preparing and determining a cPEP as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA. In view of the above, it is understood that in another embodiment, the invention relates to the method of preparing and determining a cPEP as described above, said cPEP: having a size ranging from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a. a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein; b. a step of determining within this mRNA the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein; vs. a step of determining within this nucleic acid sequence naturally translated in said plant cell a non-naturally translated fragment thereof, said non-naturally translated fragment:
- ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , - having a size of 3n nucleotides, n being between 4 and 70, in particular n being between 4 and 41,
- ayant une taille inférieure à celle de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale, - having a size smaller than that of the nucleic acid sequence naturally translated in said plant cell,
- étant dépourvu du codon initiateur AUG codant une méthionine initiatrice et/ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et- being devoid of the AUG initiator codon encoding an initiator methionine and/or a STOP codon chosen from the codons: UAG, UGA and UAA, and
- étant choisi soit dans le même cadre de lecture que le cadre ouvert de lecture codant ladite protéine, soit dans un cadre de lecture décalé d’un ou deux nucléotides par rapport au cadre ouvert de lecture codant ladite protéine ; d. une étape de production du peptide codé par ledit fragment non naturellement traduit ; et e. une étape de comparaison : - being chosen either in the same reading frame as the open reading frame encoding said protein, or in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein; d. a step of producing the peptide encoded by said non-naturally translated fragment; summer. a comparison step:
- entre l’accumulation de ladite protéine dans une cellule végétale en présence dudit peptide et l’accumulation de ladite protéine dans une cellule végétale de même type en absence dudit peptide ; et/ou - between the accumulation of said protein in a plant cell in the presence of said peptide and the accumulation of said protein in a plant cell of the same type in the absence of said peptide; and or
- entre le phénotype d’une plante en présence dudit peptide et le phénotype d’une plante de même type en absence dudit peptide, dans laquelle : - between the phenotype of a plant in the presence of said peptide and the phenotype of a plant of the same type in the absence of said peptide, in which :
- une différence de la quantité de ladite protéine en présence dudit peptide par rapport à la quantité de ladite protéine en absence dudit peptide ; et/ou - a difference in the quantity of said protein in the presence of said peptide compared to the quantity of said protein in the absence of said peptide; and or
- une différence du phénotype en présence dudit peptide par rapport au phénotype en absence dudit peptide, indique que ledit peptide est un cPEP capable de moduler l’accumulation de ladite protéine dans une cellule végétale. - a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is a cPEP capable of modulating the accumulation of said protein in a plant cell.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans le même cadre de lecture que le cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans le cadre de lecture déterminé par le codon d’initiation du cadre ouvert de lecture de ladite protéine. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame. of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open frame. reading of said protein.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit cPEP est un peptide hydrophobe ou un peptide hydrophile. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit cPEP est un peptide hydrophobe. Par « peptide hydrophobe », on entend un peptide dont la séquence en acides aminés comprend plus de 50 % d’acides aminés hydrophobes. Par « plus de 50 % », on entend que la séquence en acides aminés comprend plus de 55 %, plus de 60 %, plus de 65 %, plus de 70 %, plus de 75 % ou plus de 80 % d’acides aminés hydrophobes. Par « plus de 50 % », on entend également que la séquence en acides aminés comprend au moins 51 %, au moins 56 %, au moins 61 %, au moins 66 %, au moins 71 %, au moins 76 % ou au moins 81 % d’acides aminés hydrophobes. Par « acides aminés hydrophobes », on entend les acides aminés choisis parmi : alanine (Ala / A), isoleucine (Ile / 1), leucine (Leu / L), méthionine (Met/ M), phénylalanine (Phe / F), tryptophane (Trp / W), tyrosine (Tyr/ Y) et valine (Val / V). In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or of two nucleotides 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in 3' (or of a nucleotide at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the method for preparing and determining a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5 ') or two nucleotides 3' (or one nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said cPEP is a hydrophobic peptide or a hydrophilic peptide. In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said cPEP is a hydrophobic peptide. By “hydrophobic peptide” is meant a peptide whose amino acid sequence comprises more than 50% hydrophobic amino acids. By “more than 50%”, we mean that the amino acid sequence comprises more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80% of amino acids. hydrophobic. By “more than 50%”, we also mean that the amino acid sequence comprises at least 51%, at least 56%, at least 61%, at least 66%, at least 71%, at least 76% or at least 81% hydrophobic amino acids. By “hydrophobic amino acids” is meant the amino acids chosen from: alanine (Ala / A), isoleucine (Ile / 1), leucine (Leu / L), methionine (Met / M), phenylalanine (Phe / F), tryptophan (Trp/W), tyrosine (Tyr/Y) and valine (Val/V).
En particulier, l’invention concerne également le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ledit cPEP est un peptide hydrophile. Par « peptide hydrophile », on entend un peptide dont la séquence en acides aminés comprend plus de 50 % d’acides aminés hydrophiles. Par « plus de 50 % », on entend que la séquence en acides aminés comprend plus de 55 %, plus de 60 %, plus de 65 %, plus de 70 %, plus de 75 % ou plus de 80 % d’acides aminés hydrophiles. Par « plus de 50 % », on entend également que la séquence en acides aminés comprend au moins 51 %, au moins 56 %, au moins 61 %, au moins 66 %, au moins 71 %, au moins 76 % ou au moins 81 % d’acides aminés hydrophiles. Par « acides aminés hydrophiles », on entend les acides aminés choisis parmi : acide aspartique (Asp / D), acide glutamique (Glu / E), arginine (Arg / R), asparagine (Asn / N), glutamine (Gin / Q), histidine (His I H), lysine (Lys / K), sérine (Ser / S) et thréonine (Thr / T). In particular, the invention also relates to the method for preparing and determining a cPEP as described above, in which said cPEP is a hydrophilic peptide. By “hydrophilic peptide” is meant a peptide whose amino acid sequence comprises more than 50% hydrophilic amino acids. By “more than 50%”, we mean that the amino acid sequence comprises more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80% of amino acids. hydrophilic. By “more than 50%”, we also mean that the amino acid sequence comprises at least 51%, at least 56%, at least 61%, at least 66%, at least 71%, at least 76% or at least 81% hydrophilic amino acids. By “hydrophilic amino acids”, we mean the amino acids chosen from: aspartic acid (Asp / D), glutamic acid (Glu / E), arginine (Arg / R), asparagine (Asn / N), glutamine (Gin / Q ), histidine (His I H), lysine (Lys/K), serine (Ser/S) and threonine (Thr/T).
Dans un mode de réalisation, l’invention concerne un procédé de préparation et de détermination d’un cPEP, ledit cPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés ; étant capable de moduler l’accumulation d’une protéine dans une cellule végétale ; et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, ledit procédé comprenant : a. une étape de détermination de la séquence d’acides nucléiques de l’ARN messager (ARNm) codant ladite protéine ; b. une étape de détermination au sein de cet ARNm d’une des séquences d’acides nucléiques comprenant deux parties contiguës : In one embodiment, the invention relates to a method for preparing and determining a cPEP, said cPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a. a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein; b. a step of determining within this mRNA one of the nucleic acid sequences comprising two contiguous parts:
- une partie située au sein d’une séquence d’acides nucléiques réputée non codante (/.e. non traduite naturellement, e.g. 5’IITR ou 3’IITR), et- a part located within a nucleic acid sequence deemed non-coding (/e. not naturally translated, e.g. 5’IITR or 3’IITR), and
- une partie située au sein d’une séquence d’acides nucléiques réputée codante (/.e. traduite naturellement, e.g. exon) ; c. une étape de détermination au sein de cette séquence d’acides nucléiques comprenant deux parties contiguës d’un fragment de celle-ci, ledit fragment ayant une taille de 3n nucléotides susceptible d’être traduite via le code génétique en un peptide, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 ; d. une étape de production dudit peptide ; et e. une étape de comparaison : - a part located within a nucleic acid sequence known to be coding (/e. naturally translated, e.g. exon); vs. a determination step within this nucleic acid sequence comprising two contiguous parts of a fragment thereof, said fragment having a size of 3n nucleotides capable of being translated via the genetic code into a peptide, n being included from 4 to 70, in particular n being included from 4 to 41; d. a step of producing said peptide; summer. a comparison step:
- entre l’accumulation de ladite protéine dans une cellule végétale en présence dudit peptide et l’accumulation de ladite protéine dans une cellule végétale de même type en absence dudit peptide ; et/ou - between the accumulation of said protein in a plant cell in the presence of said peptide and the accumulation of said protein in a plant cell of the same type in the absence of said peptide; and or
- entre le phénotype d’une plante en présence dudit peptide et le phénotype d’une plante de même type en absence dudit peptide, dans laquelle : - between the phenotype of a plant in the presence of said peptide and the phenotype of a plant of the same type in the absence of said peptide, in which:
- une différence de la quantité de ladite protéine en présence dudit peptide par rapport à la quantité de ladite protéine en absence dudit peptide ; et/ou - a difference in the quantity of said protein in the presence of said peptide compared to the quantity of said protein in the absence of said peptide; and or
- une différence du phénotype en présence dudit peptide par rapport au phénotype en absence dudit peptide, indique que ledit peptide est un cPEP capable de moduler l’accumulation de ladite protéine dans une cellule végétale. - a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is a cPEP capable of modulating the accumulation of said protein in a plant cell.
Selon l’invention, un cPEP peut être produit par tout type de moyens accessibles à l’homme du métier. According to the invention, a cPEP can be produced by any type of means accessible to those skilled in the art.
De manière non limitative, un cPEP peut être produit aussi bien par synthèse, que par expression recombinante dans des systèmes homologues ou hétérologues. Le cPEP ainsi produit peut ensuite être introduit dans une cellule pour moduler l’accumulation d’une protéine cible. De manière non limitative, il est également possible de produire un cPEP directement dans la cellule végétale contenant la protéine cible, en introduisant artificiellement dans celle-ci un acide nucléique (tel qu’un vecteur d’expression) codant ledit cPEP. In a non-limiting manner, a cPEP can be produced both by synthesis and by recombinant expression in homologous or heterologous systems. The cPEP thus produced can then be introduced into a cell to modulate the accumulation of a target protein. In a non-limiting manner, it is also possible to produce a cPEP directly in the plant cell containing the target protein, by artificially introducing into it a nucleic acid (such as an expression vector) encoding said cPEP.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel, à l’étape d., la production dudit peptide est réalisée par synthèse peptidique ou par expression recombinante. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which, in step d., the production of said peptide is carried out by peptide synthesis or by recombinant expression.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel, à l’étape d., la production dudit peptide est réalisée à l’aide d’un acide nucléique codant ledit peptide introduit dans une cellule. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which, in step d., the production of said peptide is carried out using an acid nucleic acid encoding said peptide introduced into a cell.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel, à l’étape e., la production dudit peptide est réalisée à l’aide d’un acide nucléique codant ledit peptide introduit dans ladite cellule végétale ou dans ladite plante. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which, in step e., the production of said peptide is carried out using an acid nucleic acid encoding said peptide introduced into said plant cell or into said plant.
Dans un mode de réalisation, l’invention concerne un procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel, à l’étape e., ledit peptide est mis en contact avec ladite cellule végétale ou dans ladite plante. In one embodiment, the invention relates to a method for preparing and determining a cPEP as described above, in which, in step e., said peptide is brought into contact with said plant cell or in said plant .
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel, à l’étape e., ledit peptide est présent dans ladite cellule végétale ou dans ladite plante suite à l’expression d’un acide nucléique codant ledit peptide dans ladite cellule végétale ou dans ladite plante. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which, in step e., said peptide is present in said plant cell or in said plant following the expression of a nucleic acid encoding said peptide in said plant cell or in said plant.
Au vu de ce qui précède, on comprend qu’un autre mode de réalisation de l’invention a pour objet le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel, à l’étape e., la présence dudit peptide dans ladite cellule végétale ou dans ladite plante résulte : In view of the above, it is understood that another embodiment of the invention relates to the method of preparing and determining a cPEP as described above, in which, in step e., the presence of said peptide in said plant cell or in said plant results:
- de l’introduction d’une séquence d’acides nucléiques codant ledit peptide et comprenant les moyens de l’exprimer ; ou - the introduction of a nucleic acid sequence encoding said peptide and comprising the means of expressing it; Or
- de l’introduction d’une séquence d’acides aminés correspondant audit peptide. - the introduction of an amino acid sequence corresponding to said peptide.
Un cPEP peut être utilisé pour moduler l’accumulation d’une protéine présente naturellement (/.e. endogène) ou non (/.e. exogène) dans ladite cellule végétale ou dans ladite plante. Une « protéine naturellement présente dans une cellule végétale ou dans une plante » correspond à une protéine endogène codée par un gène présent sur le génome de la cellule végétale ou de la plante sans qu’il y ait eu nécessité d’intervention directe ou indirecte d’un être humain. A cPEP can be used to modulate the accumulation of a protein present naturally (/e. endogenous) or not (/e. exogenous) in said plant cell or in said plant. A “protein naturally present in a plant cell or in a plant” corresponds to an endogenous protein encoded by a gene present on the genome of the plant cell or the plant without the need for direct or indirect intervention by 'a human.
Une « protéine qui n’est pas naturellement présente dans une cellule végétale ou dans une plante » correspond à une protéine exogène codée par une séquence d’acides nucléiques présente sur le génome de la cellule végétale ou de la plante qui a nécessité l’intervention d’un être humain et l’emploi de moyens connus de l’homme du métier. Une telle séquence d’acides nucléiques peut provenir de la même espèce de plante ou d’une autre espèce de plante. A “protein which is not naturally present in a plant cell or in a plant” corresponds to an exogenous protein encoded by a nucleic acid sequence present on the genome of the plant cell or plant which required the intervention of a human being and the use of means known to those skilled in the art. Such a nucleic acid sequence may come from the same plant species or from a different plant species.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est d’origine endogène dans lesdites cellules végétales ou lesdites plantes utilisées à l’étape e. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is of endogenous origin in said plant cells or said plants used in step e.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est d’origine exogène dans lesdites cellules végétales ou lesdites plantes utilisées à l’étape e., lesdites cellules végétales ou lesdites plantes utilisées à l’étape e. comprenant alors une séquence d’acides nucléiques permettant l’expression de ladite protéine. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is of exogenous origin in said plant cells or said plants used in step e., said plant cells or said plants used in step e. then comprising a nucleic acid sequence allowing the expression of said protein.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel l’accumulation de ladite protéine est déterminée via la mise en œuvre d’une technique choisie parmi : le Western blot, la mesure de l’activité enzymatique, la spectrométrie de masse et la fusion traductionnelle. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel l’accumulation de ladite protéine est déterminée via la mise en œuvre d’un Western blot. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blot, measurement of enzyme activity, mass spectrometry and translational fusion. In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
Les Inventeurs ont constaté de manière surprenante que l’utilisation des cPEPs permet de modifier les phénotypes d’une plante visibles à l’échelle macroscopique. Il est donc tout à fait possible d’utiliser ces derniers pour affirmer (ou infirmer) que le peptide déterminé aux étapes a., b. et c., et éventuellement produit à l’étape d. est un cPEP (ou non). C’est d’ailleurs ce que permet l’alternative dite de comparaison phénotypique mise en œuvre à l’étape e. Par exemple, si le peptide déterminé sur l’ARNm d’une protéine impliquée dans la taille de la tige d’une plante provoque une augmentation, ou une diminution, de la taille de la tige d’une plante traitée avec ce dernier par rapport à une plante non traitée, cela signifie que ledit peptide est un cPEP capable de moduler l’accumulation de ladite protéine dans la taille de la tige. Dans l’invention, le terme « plante » fait référence de manière générale : à un ensemble de cellules végétales organisé en tout ou partie d’une plante quel que soit son stade de développement (y compris la plante sous forme de graine ou de jeune pousse) ; à un ou plusieurs organes de la plante (comme par exemple les feuilles, les racines, la tige, les fleurs) ; à une ou plusieurs cellules de la plante ; ou encore à un amas de cellules de la plante {e.g. un cal). The inventors have surprisingly noted that the use of cPEPs makes it possible to modify the phenotypes of a plant visible on a macroscopic scale. It is therefore entirely possible to use the latter to affirm (or refute) that the peptide determined in steps a., b. and c., and optionally produced in step d. is a cPEP (or not). This is also what the so-called phenotypic comparison alternative implemented in step e allows. For example, if the peptide determined on the mRNA of a protein involved in the size of the stem of a plant causes an increase, or a decrease, in the size of the stem of a plant treated with the latter compared to an untreated plant, this means that said peptide is a cPEP capable of modulating the accumulation of said protein in the size of the stem. In the invention, the term "plant" refers generally: to a set of plant cells organized in all or part of a plant whatever its stage of development (including the plant in the form of seed or young grows); to one or more organs of the plant (such as leaves, roots, stem, flowers); to one or more cells of the plant; or even a mass of plant cells {eg a callus).
Dans l’invention, le terme « phénotype » désigne de manière non limitative les caractères visibles à l’échelle macroscopique tel que le nombre de racines latérales, le nombre de feuilles, la taille de la tige, la durée de floraison et la résistance au stress. Dans un mode de réalisation, l’invention concerne par conséquent le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est impliquée dans au moins un phénotype de plante choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In the invention, the term "phenotype" designates in a non-limiting manner the characters visible on a macroscopic scale such as the number of lateral roots, the number of leaves, the size of the stem, the duration of flowering and the resistance to stress. In one embodiment, the invention therefore relates to the method for preparing and determining a cPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Selon l’invention, une protéine est « impliquée dans un phénotype » si une modification de l’accumulation de celle-ci est associée à une modification dudit phénotype. En d’autres termes, une protéine est impliquée dans un phénotype si elle intervient dans le(s) caractère(s) correspondant audit phénotype. According to the invention, a protein is “involved in a phenotype” if a modification of the accumulation thereof is associated with a modification of said phenotype. In other words, a protein is involved in a phenotype if it intervenes in the character(s) corresponding to said phenotype.
Au vu de ce qui précède, on comprend qu’un objet de l’invention est le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel le phénotype observé à l’étape e. est choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In view of the above, it is understood that an object of the invention is the process for preparing and determining a cPEP as described above, in which the phenotype observed in step e. is chosen from: the size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, ledit cPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés ; étant capable de moduler l’accumulation d’une protéine dans une cellule végétale ; et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, et dans lequel ladite cellule végétale (/.e. celle dans laquelle on souhaite moduler l’accumulation d’une protéine) appartient à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, said cPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, and in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) belongs to a chosen plant species among: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel lesdites cellules végétales ou lesdites plantes utilisées à l’étape e. appartiennent à : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said plant cells or said plants used in step e. belong to: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora , Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite cellule végétale (/.e. celle dans laquelle on souhaite moduler l’accumulation d’une protéine) est une cellule d’une algue. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) is a cell of an algae.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel lesdites cellules végétales ou lesdites plantes utilisées à l’étape e appartiennent à une algue. In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said plant cells or said plants used in step e belong to an alga.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi : Aae15 (Acyl-activating enzyme 15), Aae16 (AMP-dependent synthetase and ligase family protein), Abcg11 (White-brown complex-like protein), Abdcg34 (ABC transporter G family member 34), Acc1 (Acetyl-CoA Carboxylase), Agb1 (GTP binding protein beta 1), Als (Acetolactate synthase (chloroplastic)), Anac076 (NAC domain-containing protein 76), Apg9 (Autophagy 9), Arlbl (GTP-binding protein 1), Arr1 (Two-component response regulator ARR1), Arr5 (Two-component response regulator ARR5), Arr6 (Two- component response regulator ARR6), At59 (Pedate lyase family protein), Bak1 (Brassinosteroid insensitive 1 -associated receptor kinase 1), Bccpl (Acetyl-CoA Carboxylase (chloroplastic) subunit 1), Bccp2 (Acetyl-CoA Carboxylase (chloroplastic) subunit 2), Bril (Brassinosteroid insensitive 1), Bzo2h3 (bZIP transcription factor family protein), Cesa6 (Cellulose synthase A catalytic subunit 6), Cipk3 (CBL-intera ing protein kinase 3), Cks1 (Cyclin-dependent kinases regulatory subunit 1), Cobl8 (COBRA-like protein 8 precursor), Coil (Coronatine-insensitive protein 1), Cpk3 (Calcium-dependent protein kinase 3), Crk34 (Cysteine-rich receptor-like protein kinase 34), Cyp705a18 (Cytochrome P450, family 705, subfamily A, polypeptide 18), Cyp71b26 (Cytochrome P450, family 71, subfamily B, polypeptide 26), Cyp78a8 (Cytochrome P450, family 78, subfamily A, polypeptide 8), Cyp97b3 (Cytochrome P450, family 97, subfamily B, polypeptide 3), Dell (Endoribonuclease Dicer homolog 1), Dur3 (Urea-proton symporter DUR3), Ein2 (Ethylene-insensitive protein 2), Emb 175 (Pentatricopeptide repeat-containing protein), Emb2726 (Elongation factor Ts family protein), Emb9 (Di hydrofolate synthetase), Epsps (5-enolpyruvylshikimate-3-phosphate (chloroplastic)), Fnr1 (Ferredoxin-NADP[+]-oxidoreductase 1), Fve (Transducin family protein / WD-40 repeat family protein), Ga2ox7 (Gibberellin 2-beta-dioxygenase 7), Gape (Glyceraldehyde-3-phosphate dehydrogenase), Gcn2 (ABC transporter family protein), Gdi2 (Guanosine nucleotide diphosphate dissociation inhibitor 2), Gln2 (Glutamine synthetase (chloroplastic)), Gsl3 (Callose synthase 2), Hag5 (Histone acetyltransferase of the MYST family 2), Hda18 (Histone deacetylase 18), Hexol (Beta-hexosaminidase 1), Hppd (4- hydroxyphenyl-pyruvate-dioxygenase), Hsl1 (B3 domain-containing transcription repressor VAL2), Iaa31 (lndole-3-acetic acid inducible 31), Iqd28 (IQ-domain 28), Jac1 (J-domain protein required for chloroplast accumulation response 1), Jar1 (Jasmonoyl-L-amino acid synthetase), Kp1 (Kinesin-like protein 1), Lrx2 (Leucine-rich repeat/extensin 2), Mapkkk3 (Mitogen-activated protein kinase kinase kinase 3), Mapkkk5 (Mitogen-activated protein kinase kinase kinase 5), Mfp2 (Multifunctional protein 2), Mrb1 (Transmembrane protein, putative (DUF3537)), Nsp1 (Nodulation signaling pathway 1), Pds (Phytoene desaturase (chloroplastic)), Pen3 (Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein- tyrosine-phosphatase), Phyb (Phytochrome B), Pif3 (Phytochrome interacting factor 3), Pizza (Brassinosteroid-related acyltransferase 1), Ppoxl (Protoporphyrinogen oxidase (chloroplastic) 1), Ppox2 (Protoporphyrinogen oxidase (chloroplastic) 2), Prp39 (Tetratricopeptide repeat (TPR)-like superfamily protein), PsbA (Photosystem II D1 protein), Pskrl (Phytosulfokin receptor 1), Rd21 (Granulin repeat cysteine protease family protein), Ringl (RING/U-box superfamily protein), Rosi (DNA glycosylase/AP lyase ROS1), Rpt4a (26S proteasome regulatory subunit 10B homolog A), Sfr6 (Mediator of RNA polymerase II transcription subunit 16), Shr (Protein SHORT-ROOT), Shy2 (Auxin-responsive protein IAA3), Ski (EIN2-like protein, nramp transporter), Sps1 (Sucrose phosphate synthase 2F), Spt (Transcription factor SPATULA), Stn8 (Serine/threonine-protein kinase), Tap46 (PP2A regulatory subunit TAP46), Topp6 (Serine/threonine-protein phosphatase PP1 isozyme 7), TubB6 (Tubulin), TubB8 (Tubulin), Ubala (RNA-binding (RRM/RBD/RNP motifs) family protein), Vim3 (E3 ubiquitin-protein ligase), Sgr1 (Magnesium dechelatase), Abi5 (Abscisic acid (ABA)-insensitive 5), Hsp101 (Heat shock protein 101), Rh10 (ATP-dependent RNA helicase) et Wus (WUSCHEL). In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene chosen from: Aae15 (Acyl-activating enzyme 15), Aae16 (AMP -dependent synthetase and ligase family protein), Abcg11 (White-brown complex-like protein), Abdcg34 (ABC transporter G family member 34), Acc1 (Acetyl-CoA Carboxylase), Agb1 (GTP binding protein beta 1), Als (Acetolactate synthase (chloroplastic)), Anac076 (NAC domain-containing protein 76), Apg9 (Autophagy 9), Arlbl (GTP-binding protein 1), Arr1 (Two-component response regulator ARR1), Arr5 (Two-component response regulator ARR5) , Arr6 (Two-component response regulator ARR6), At59 (Pedate lyase family protein), Bak1 (Brassinosteroid insensitive 1 -associated receptor kinase 1), Bccpl (Acetyl-CoA Carboxylase (chloroplastic) subunit 1), Bccp2 (Acetyl-CoA Carboxylase (chloroplastic) subunit 2), Bril (Brassinosteroid insensitive 1), Bzo2h3 (bZIP transcription factor family protein), Cesa6 (Cellulose synthase A catalytic subunit 6), Cipk3 (CBL-intera ing protein kinase 3), Cks1 (Cyclin-dependent kinases regulatory subunit 1), Cobl8 (COBRA-like protein 8 precursor), Coil (Coronatine-insensitive protein 1), Cpk3 (Calcium-dependent protein kinase 3), Crk34 (Cysteine-rich receptor-like protein kinase 34), Cyp705a18 (Cytochrome P450, family 705, subfamily A, polypeptide 18), Cyp71b26 (Cytochrome P450, family 71, subfamily B, polypeptide 26), Cyp78a8 (Cytochrome P450, family 78, subfamily A, polypeptide 8), Cyp97b3 (Cytochrome P450, family 97, subfamily B, polypeptide 3), Dell (Endoribonuclease Dicer homolog 1), Dur3 (Urea-proton symporter DUR3) , Ein2 (Ethylene-insensitive protein 2), Emb 175 (Pentatricopeptide repeat-containing protein), Emb2726 (Elongation factor Ts family protein), Emb9 (Di hydrofolate synthetase), Epsps (5-enolpyruvylshikimate-3-phosphate (chloroplastic)), Fnr1 (Ferredoxin-NADP[+]-oxidoreductase 1), Fve (Transducin family protein / WD-40 repeat family protein), Ga2ox7 (Gibberellin 2-beta-dioxygenase 7), Gape (Glyceraldehyde-3-phosphate dehydrogenase), Gcn2 ( ABC transporter family protein), Gdi2 (Guanosine nucleotide diphosphate dissociation inhibitor 2), Gln2 (Glutamine synthetase (chloroplastic)), Gsl3 (Callose synthase 2), Hag5 (Histone acetyltransferase of the MYST family 2), Hda18 (Histone deacetylase 18), Hexol (Beta-hexosaminidase 1), Hppd (4-hydroxyphenyl-pyruvate-dioxygenase), Hsl1 (B3 domain-containing transcription repressor VAL2), Iaa31 (lndole-3-acetic acid inducible 31), Iqd28 (IQ-domain 28), Jac1 (J-domain protein required for chloroplast accumulation response 1), Jar1 (Jasmonoyl-L-amino acid synthetase), Kp1 (Kinesin-like protein 1), Lrx2 (Leucine-rich repeat/extensin 2), Mapkkk3 (Mitogen-activated protein kinase kinase kinase kinase 3), Mapkkk5 (Mitogen-activated protein kinase kinase kinase 5), Mfp2 (Multifunctional protein 2), Mrb1 (Transmembrane protein, putative (DUF3537)), Nsp1 (Nodulation signaling pathway 1), Pds (Phytoene desaturase ( chloroplastic)), Pen3 (Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein-tyrosine-phosphatase), Phyb (Phytochrome B), Pif3 (Phytochrome interacting factor 3), Pizza (Brassinosteroid-related acyltransferase 1), Ppoxl ( Protoporphyrinogen oxidase (chloroplastic) 1), Ppox2 (Protoporphyrinogen oxidase (chloroplastic) 2), Prp39 (Tetratricopeptide repeat (TPR)-like superfamily protein), PsbA (Photosystem II D1 protein), Pskrl (Phytosulfokin receptor 1), Rd21 (Granulin repeat cysteine protease family protein), Ringl (RING/U-box superfamily protein), Rosi (DNA glycosylase/AP lyase ROS1), Rpt4a (26S proteasome regulatory subunit 10B homolog A), Sfr6 (Mediator of RNA polymerase II transcription subunit 16), Shr (SHORT-ROOT protein), Shy2 (Auxin-responsive protein IAA3), Ski (EIN2-like protein, nramp transporter), Sps1 (Sucrose phosphate synthase 2F), Spt (Transcription factor SPATULA), Stn8 (Serine/threonine-protein kinase), Tap46 (PP2A regulatory subunit TAP46), Topp6 (Serine/threonine-protein phosphatase PP1 isozyme 7), TubB6 (Tubulin), TubB8 (Tubulin), Ubala (RNA-binding (RRM/RBD/RNP motifs) family protein) , Vim3 (E3 ubiquitin-protein ligase), Sgr1 (Magnesium dechelatase), Abi5 (Abscisic acid (ABA)-insensitive 5), Hsp101 (Heat shock protein 101), Rh10 (ATP-dependent RNA helicase) and Wus (WUSCHEL).
En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi : Cpk3, Dell et Nsp1. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Cpk3. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Dell. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Nsp1. In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene chosen from: Cpk3, Dell and Nsp1. In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by the Cpk3 gene. In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by the Dell gene. In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by the Nsp1 gene.
Les gènes Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 et l/l/us font références aux protéines indiquées entre parenthèses. Bien entendu, l’invention concerne également des gènes homologues et/ou similaires susceptibles de porter des dénominations différentes. Par exemple, chez A. thaliana le gène Gsl3 codant pour la callose synthase 2 s’appelle également Cals2. The genes Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi , Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 and l/l/us refer to proteins indicated in parentheses. Of course, the invention also relates to homologous and/or similar genes likely to bear different names. For example, in A. thaliana the Gsl3 gene encoding callose synthase 2 is also called Cals2.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NO : 1 (ORF de la protéine Aae15, A. thaliana), SEQ ID NO : 2 (ORF de la protéine Aae16, A. thaliana), SEQ ID NO : 3 (ORF de la protéine abcg11, A. thaliana), SEQ ID NO : 4 (ORF de la protéine Abdcg34, A. thaliana), SEQ ID NO : 5 (ORF de la protéine Acc1, A. thaliana), SEQ ID NO : 6 (ORF de la protéine Agb1 , A. thaliana), SEQ ID NO : 7 (ORF de la protéine Als, A. thaliana), SEQ ID NO : 8 (ORF de la protéine Anac076, A. thaliana), SEQ ID NO : 9 (ORF de la protéine Apg9, A. thaliana), SEQ ID NO : 10 (ORF de la protéine Arlbl, A. thaliana), SEQ ID NO : 11 (ORF de la protéine Arr1, A. thaliana), SEQ ID NO : 12 (ORF de la protéine Arr5, A. thaliana), SEQ ID NO : 13 (ORF de la protéine Arr6, A. thaliana), SEQ ID NO : 14 (ORF de la protéine At59, A. thaliana), SEQ ID NO : 15 (ORF de la protéine Bak1, A. thaliana), SEQ ID NO : 16 (ORF de la protéine Bccpl , A. thaliana), SEQ ID NO : 17 (ORF de la protéine Bccp2, A. thaliana), SEQ ID NO : 18 (ORF de la protéine Bri1 , A. thaliana), SEQ ID NO : 19 (ORF de la protéine Bzo2h3, A. thaliana), SEQ ID NO : 20 (ORF de la protéine Cesa6, A. thaliana), SEQ ID NO : 21 (ORF de la protéine Cipk3, A. thaliana), SEQ ID NO : 22 (ORF de la protéine Cks1, A thaliana), SEQ ID NO : 23 (ORF de la protéine Cobl8, A thaliana), SEQ ID NO : 24 (ORF de la protéine Coi1, A. thaliana), SEQ ID NO : 25 (ORF de la protéine Coil, A. thaliana), SEQ ID NO : 26 (ORF de la protéine Cpk3, A. thaliana), SEQ ID NO : 27 (ORF de la protéine Cpk3, A. hypochondriacus), SEQ ID NO : 28 (ORF de la protéine Cpk3, B. distachyori), SEQ ID NO : 29 (ORF de la protéine Cpk3, B. distachyori), SEQ ID NO : 30 (ORF de la protéine Cpk3, G. max), SEQ ID NO : 31 (ORF de la protéine Cpk3, G. max), SEQ ID NO : 32 (ORF de la protéine Cpk3, G. max), SEQ ID NO : 33 (ORF de la protéine Cpk3, G. max), SEQ ID NO : 34 (ORF de la protéine Cpk3, O. sativa), SEQ ID NO : 35 (ORF de la protéine Cpk3, O. sativa), SEQ ID NO : 36 (ORF de la protéine Cpk3, S. lycopersicum), SEQ ID NO : 37 (ORF de la protéine Cpk3, Z. mays), SEQ ID NO : 38 (ORF de la protéine Cpk3, Z. mays), SEQ ID NO : 39 (ORF de la protéine Cpk3, Z. mays), SEQ ID NO : 40 (ORF de la protéine Cpk3, B. rapa), SEQ ID NO : 41 (ORF de la protéine Cpk3, B. rapa), SEQ ID NO : 42 (ORF de la protéine Cpk3, H. vulgare), SEQ ID NO : 43 (ORF de la protéine Cpk3, H. vulgare), SEQ ID NO : 44 (ORF de la protéine Cpk3, S. tuberosum), SEQ ID NO : 45 (ORF de la protéine Cpk3, A. palmeri), SEQ ID NO : 46 (ORF de la protéine Cpk3, M. truncatula), SEQ ID NO : 47 (ORF de la protéine Cpk3, M. truncatula), SEQ ID NO : 48 (ORF de la protéine Cpk3, T. aestivum), SEQ ID NO : 49 (ORF de la protéine Cpk3, T. aestivum), SEQ ID NO : 50 (ORF de la protéine Cpk3, T. aestivum), SEQ ID NO : 51 (ORF de la protéine Cpk3, T. aestivum), SEQ ID NO : 52 (ORF de la protéine Cpk3, L. perenne), SEQ ID NO : 53 (ORF de la protéine Cpk3, L. perenne), SEQ ID NO : 54 (ORF de la protéine Cpk3, L. perenne), SEQ ID NO : 55 (ORF de la protéine Cpk3, L. perenne), SEQ ID NO : 56 (ORF de la protéine Crk34, A. thaliana), SEQ ID NO : 57 (ORF de la protéine Cyp705a18, A. thaliana), SEQ ID NO : 58 (ORF de la protéine Cyp71b26, A. thaliana), SEQ ID NO : 59 (ORF de la protéine Cyp78a8, A. thaliana), SEQ ID NO : 60 (ORF de la protéine Cyp97b3, A. thaliana), SEQ ID NO : 61 (ORF de la protéine Dell, A. thaliana), SEQ ID NO : 62 (ORF de la protéine Dell, A. thaliana), SEQ ID NO : 63 (ORF de la protéine Dell , A. hypochondriacus), SEQ ID NO : 64 (ORF de la protéine Dell, B. distachyon), SEQ ID NO : 65 (ORF de la protéine Dell, G. max), SEQ ID NO : 66 (ORF de la protéine Dell, G. max), SEQ ID NO : 67 (ORF de la protéine Dell, O. sativa), SEQ ID NO : 68 (ORF de la protéine Dell , S. lycopersicum), SEQ ID NO : 69 (ORF de la protéine Dell , Z. mays), SEQ ID NO : 70 (ORF de la protéine Dell , B. rapa), SEQ ID NO : 71 (ORF de la protéine Dell, H. vulgare), SEQ ID NO : 72 (ORF de la protéine Dell, S. tuberosum), SEQ ID NO : 73 (ORF de la protéine Dell, M. truncatula), SEQ ID NO : 74 (ORF de la protéine Dell, T. aestivum), SEQ ID NO : 75 (ORF de la protéine Dell, T. aestivum), SEQ ID NO : 76 (ORF de la protéine Dell, T. aestivum), SEQ ID NO : 77 (ORF de la protéine Dell, T. aestivum), SEQ ID NO : 78 (ORF de la protéine Dell , L. perenne), SEQ ID NO : 79 (ORF de la protéine Dell , L. perenne), SEQ ID NO : 80 (ORF de la protéine Dur3, A thaliana), SEQ ID NO : 81 (ORF de la protéine Ein2, Æ thaliana), SEQ ID NO : 82 (ORF de la protéine Emb175, A. thaliana), SEQ ID NO : 83 (ORF de la protéine Emb2726, A. thaliana), SEQ ID NO : 84 (ORF de la protéine Emb9, A. thaliana), SEQ ID NO : 85 (ORF de la protéine Epsps, A. thaliana), SEQ ID NO : 86 (ORF de la protéine Fnr1 , A. thaliana), SEQ ID NO : 87 (ORF de la protéine Fve, A. thaliana), SEQ ID NO : 88 (ORF de la protéine Ga2ox7, A. thaliana), SEQ ID NO : 89 (ORF de la protéine Gape, N. benthamiana), SEQ ID NO : 90 (ORF de la protéine Gcn2, A. thaliana), SEQ ID NO : 91 (ORF de la protéine Gdi2, A. thaliana), SEQ ID NO : 92 (ORF de la protéine Gln2, A. thaliana), SEQ ID NO : 93 (ORF de la protéine Gsl3, A. thaliana), SEQ ID NO : 94 (ORF de la protéine Hag5, A. thaliana), SEQ ID NO : 95 (ORF de la protéine Hda18, A. thaliana), SEQ ID NO : 96 (ORF de la protéine Hexol , A. thaliana), SEQ ID NO : 97 (ORF de la protéine Hppd, A. thaliana), SEQ ID NO : 98 (ORF de la protéine Hsl1, A. thaliana), SEQ ID NO : 99 (ORF de la protéine Iaa31, A. thaliana), SEQ ID NO : 100 (ORF de la protéine Iqd28, A. thaliana), SEQ ID NO : 101 (ORF de la protéine Jac1 , A. thaliana), SEQ ID NO : 102 (ORF de la protéine Jar1 , A. thaliana), SEQ ID NO : 103 (ORF de la protéine Kp1 , A. thaliana), SEQ ID NO : 104 (ORF de la protéine Lrx2, A. thaliana), SEQ ID NO : 105 (ORF de la protéine Mapkkk3, A. thaliana), SEQ ID NO : 106 (ORF de la protéine Mapkkk5, A. thaliana), SEQ ID NO : 107 (ORF de la protéine Mfp2, A. thaliana), SEQ ID NO : 108 (ORF de la protéine Mrb1, A. thaliana), SEQ ID NO : 109 (ORF de la protéine Nsp1, M. truncatula), SEQ ID NO : 110 (ORF de la protéine Nsp1, A. thaliana), SEQ ID NO : 111 (ORF de la protéine Nsp1 , B. distachyon), SEQ ID NO : 112 (ORF de la protéine Nsp1, G. max), SEQ ID NO : 113 (ORF de la protéine Nsp1, G. max), SEQ ID NO : 114 (ORF de la protéine Nsp1, O. sativa), SEQ ID NO : 115 (ORF de la protéine Nsp1 , S. lycopersicum), SEQ ID NO : 116 (ORF de la protéine Nsp1, S. lycopersicum), SEQ ID NO : 117 (ORF de la protéine Nsp1, Z. mays), SEQ ID NO : 118 (ORF de la protéine Nsp1, Z. mays), SEQ ID NO : 119 (ORF de la protéine Nsp1, Z. mays), SEQ ID NO : 120 (ORF de la protéine Nsp1 , Z. mays), SEQ ID NO : 121 (ORF de la protéine Nsp1 , B. rapa), SEQ ID NO : 122 (ORF de la protéine Nsp1 , H. vulgare), SEQ ID NO : 123 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 124 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 125 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 126 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 127 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 128 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 129 (ORF de la protéine Nsp1 , S. tuberosum), SEQ ID NO : 130 (ORF de la protéine Nsp1, S. tuberosum), SEQ ID NO : 131 (ORF de la protéine Nsp1, T. aestivum), SEQ ID NO : 132 (ORF de la protéine Nsp1 , T. aestivum), SEQ ID NO : 133 (ORF de la protéine Nsp1 , L. perenne), SEQ ID NO : 134 (ORF de la protéine Nsp1, L. perenne), SEQ ID NO : 135 (ORF de la protéine Pds, A. thaliana), SEQ ID NO : 136 (ORF de la protéine Pen3, A. thaliana), SEQ ID NO : 137 (ORF de la protéine Phyb, A. thaliana), SEQ ID NO : 138 (ORF de la protéine Pif3, A. thaliana), SEQ ID NO : 139 (ORF de la protéine Pizza, A thaliana), SEQ ID NO : 140 (ORF de la protéine Ppoxl, A thaliana), SEQ ID NO : 141 (ORF de la protéine Ppox2, A. thaliana), SEQ ID NO : 142 (ORF de la protéine Prp39, A. thaliana), SEQ ID NO : 143 (ORF de la protéine PsbA, A. thaliana), SEQ ID NO : 144 (ORF de la protéine Pskrl, A. thaliana), SEQ ID NO : 145 (ORF de la protéine Rd21, A. thaliana), SEQ ID NO : 146 (ORF de la protéine Ringl, A. thaliana), SEQ ID NO : 147 (ORF de la protéine Rosi , A. thaliana), SEQ ID NO : 148 (ORF de la protéine Rpt4a, A. thaliana), SEQ ID NO : 149 (ORF de la protéine Sfr6, A. thaliana), SEQ ID NO : 150 (ORF de la protéine Shr, A. thaliana), SEQ ID NO : 151 (ORF de la protéine Shy2, A. thaliana), SEQ ID NO : 152 (ORF de la protéine Ski, M. truncatula), SEQ ID NO : 153 (ORF de la protéine Sps1 , A. thaliana), SEQ ID NO : 154 (ORF de la protéine Spt, A. thaliana), SEQ ID NO : 155 (ORF de la protéine Stn8, A. thaliana), SEQ ID NO : 156 (ORF de la protéine Tap46, A. thaliana), SEQ ID NO : 157 (ORF de la protéine Topp6, A. thaliana), SEQ ID NO : 158 (ORF de la protéine TubB6, A. thaliana), SEQ ID NO : 159 (ORF de la protéine TubB8, A. thaliana), SEQ ID NO : 160 (ORF de la protéine llbala, A. thaliana), SEQ ID NO : 161 (ORF de la protéine Vim3, A. thaliana), SEQ ID NO : 381 (ORF de la protéine Sgr1 , A. thaliana), SEQ ID NO : 382 (ORF de la protéine Abi5, A. thaliana), SEQ ID NO : 383 (ORF de la protéine Hsp101, A. thaliana), SEQ ID NO : 384 (ORF de la protéine Rh10, M. truncatula) et SEQ ID NO : 385 (ORF de la protéine Wus, A. thaliana). In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity. , preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NO: 1 (ORF of the Aae15 protein, A. thaliana), SEQ ID NO: 2 (ORF of the Aae16 protein, A. thaliana), SEQ ID NO: 3 (ORF of the abcg11 protein, A. thaliana), SEQ ID NO: 4 (ORF of the Abdcg34 protein, A. thaliana), SEQ ID NO: 5 (ORF of the Acc1 protein, A . thaliana), SEQ ID NO: 6 (ORF of the Agb1 protein, A. thaliana), SEQ ID NO: 7 (ORF of the Als protein, A. thaliana), SEQ ID NO: 8 (ORF of the Anac076 protein, A. thaliana), SEQ ID NO: 9 (ORF of the Apg9 protein, A. thaliana), SEQ ID NO: 10 (ORF of the Arlbl protein, A. thaliana), SEQ ID NO: 11 (ORF of the Arr1 protein , A. thaliana), SEQ ID NO: 12 (ORF of the Arr5 protein, A. thaliana), SEQ ID NO: 13 (ORF of the Arr6 protein, A. thaliana), SEQ ID NO: 14 (ORF of the protein At59, A. thaliana), SEQ ID NO: 15 (ORF of the Bak1 protein, A. thaliana), SEQ ID NO: 16 (ORF of the Bccpl protein, A. thaliana), SEQ ID NO: 17 (ORF of the Bccp2 protein, A. thaliana), SEQ ID NO: 18 (ORF of the Bri1 protein, A. thaliana), SEQ ID NO: 19 (ORF of the Bzo2h3 protein, A. thaliana), SEQ ID NO: 20 (ORF of the Cesa6 protein, A. thaliana), SEQ ID NO: 21 (ORF of the Cipk3 protein, A. thaliana), SEQ ID NO: 22 (ORF of the Cks1 protein, A thaliana), SEQ ID NO: 23 (ORF of the Cobl8 protein, A thaliana), SEQ ID NO: 24 (ORF of the Coi1 protein, A. thaliana), SEQ ID NO: 25 ( ORF of the Coil protein, A. thaliana), SEQ ID NO: 26 (ORF of the Cpk3 protein, A. thaliana), SEQ ID NO: 27 (ORF of the Cpk3 protein, A. hypochondriacus), SEQ ID NO: 28 (ORF of the Cpk3 protein, B. distachyori), SEQ ID NO: 29 (ORF of the Cpk3 protein, B. distachyori), SEQ ID NO: 30 (ORF of the Cpk3 protein, G. max), SEQ ID NO: 31 (ORF of the Cpk3 protein, G. max), SEQ ID NO: 32 (ORF of the Cpk3 protein, G. max), SEQ ID NO: 33 (ORF of the Cpk3 protein, G. max), SEQ ID NO : 34 (ORF of the Cpk3 protein, O. sativa), SEQ ID NO: 35 (ORF of the Cpk3 protein, O. sativa), SEQ ID NO: 36 (ORF of the Cpk3 protein, S. lycopersicum), SEQ ID NO: 37 (ORF of the Cpk3 protein, Z. mays), SEQ ID NO: 38 (ORF of the Cpk3 protein, Z. mays), SEQ ID NO: 39 (ORF of the Cpk3 protein, Z. mays), SEQ ID NO: 40 (ORF of the Cpk3 protein, B. rapa), SEQ ID NO: 41 (ORF of the Cpk3 protein, B. rapa), SEQ ID NO: 42 (ORF of the Cpk3 protein, H. vulgare), SEQ ID NO: 43 (ORF of the Cpk3 protein, H. vulgare), SEQ ID NO: 44 (ORF of the Cpk3 protein, S. tuberosum), SEQ ID NO: 45 (ORF of the Cpk3 protein, A. palmeri) , SEQ ID NO: 46 (ORF of the Cpk3 protein, M. truncatula), SEQ ID NO: 47 (ORF of the Cpk3 protein, M. truncatula), SEQ ID NO: 48 (ORF of the Cpk3 protein, T. aestivum ), SEQ ID NO: 49 (ORF of the Cpk3 protein, T. aestivum), SEQ ID NO: 50 (ORF of the Cpk3 protein, T. aestivum), SEQ ID NO: 51 (ORF of the Cpk3 protein, T. aestivum), SEQ ID NO: 52 (ORF of the Cpk3 protein, L. perenne), SEQ ID NO: 53 (ORF of the Cpk3 protein, L. perenne), SEQ ID NO: 54 (ORF of the Cpk3 protein, L . perenne), SEQ ID NO: 55 (ORF of the Cpk3 protein, L. perenne), SEQ ID NO: 56 (ORF of the Crk34 protein, A. thaliana), SEQ ID NO: 57 (ORF of the Cyp705a18 protein, A. thaliana), SEQ ID NO: 58 (ORF of the Cyp71b26 protein, A. thaliana), SEQ ID NO: 59 (ORF of the Cyp78a8 protein, A. thaliana), SEQ ID NO: 60 (ORF of the Cyp97b3 protein , A. thaliana), SEQ ID NO: 61 (ORF of the Dell protein, A. thaliana), SEQ ID NO: 62 (ORF of the Dell protein, A. thaliana), SEQ ID NO: 63 (ORF of the protein Dell, A. hypochondriacus), SEQ ID NO: 64 (ORF of the Dell protein, B. distachyon), SEQ ID NO: 65 (ORF of the Dell protein, G. max), SEQ ID NO: 66 (ORF of the Dell protein, G. max), SEQ ID NO: 67 (ORF of the Dell protein, O. sativa), SEQ ID NO: 68 (ORF of the Dell protein, S. lycopersicum), SEQ ID NO: 69 (ORF of the Dell protein, Z. mays), SEQ ID NO: 70 (ORF of the Dell protein, B. rapa), SEQ ID NO: 71 (ORF of the Dell protein, H. vulgare), SEQ ID NO: 72 (ORF of the Dell protein, S. tuberosum), SEQ ID NO: 73 (ORF of the Dell protein, M. truncatula), SEQ ID NO: 74 (ORF of the Dell protein, T. aestivum), SEQ ID NO: 75 ( ORF of the Dell protein, T. aestivum), SEQ ID NO: 76 (ORF of the Dell protein, T. aestivum), SEQ ID NO: 77 (ORF of the Dell protein, T. aestivum), SEQ ID NO: 78 (ORF of the Dell protein, L. perenne), SEQ ID NO: 79 (ORF of the Dell protein, L. perenne), SEQ ID NO: 80 (ORF of the Dur3 protein, A thaliana), SEQ ID NO: 81 (ORF of the Ein2 protein, A. thaliana), SEQ ID NO: 82 (ORF of the Emb175 protein, A. thaliana), SEQ ID NO: 83 (ORF of the Emb2726 protein, A. thaliana), SEQ ID NO: 84 (ORF of the Emb9 protein, A. thaliana), SEQ ID NO: 85 (ORF of the Epsps protein, A. thaliana), SEQ ID NO: 86 (ORF of the Fnr1 protein, A. thaliana) , SEQ ID NO: 87 (ORF of the Fve protein, A. thaliana), SEQ ID NO: 88 (ORF of the Ga2ox7 protein, A. thaliana), SEQ ID NO: 89 (ORF of the Gape protein, N. benthamiana ), SEQ ID NO: 90 (ORF of the Gcn2 protein, A. thaliana), SEQ ID NO: 91 (ORF of the Gdi2 protein, A. thaliana), SEQ ID NO: 92 (ORF of the Gln2 protein, A. thaliana), SEQ ID NO: 93 (ORF of the Gsl3 protein, A. thaliana), SEQ ID NO: 94 (ORF of the Hag5 protein, A. thaliana), SEQ ID NO: 95 (ORF of the Hda18 protein, A . thaliana), SEQ ID NO: 96 (ORF of the Hexol protein, A. thaliana), SEQ ID NO: 97 (ORF of the Hppd protein, A. thaliana), SEQ ID NO: 98 (ORF of the Hsl1 protein, A. thaliana), SEQ ID NO: 99 (ORF of the Iaa31 protein, A. thaliana), SEQ ID NO: 100 (ORF of the Iqd28 protein, A. thaliana), SEQ ID NO: 101 (ORF of the Jac1 protein , A. thaliana), SEQ ID NO: 102 (ORF of the Jar1 protein, A. thaliana), SEQ ID NO: 103 (ORF of the Kp1 protein, A. thaliana), SEQ ID NO: 104 (ORF of the protein Lrx2, A. thaliana), SEQ ID NO: 105 (ORF of the Mapkkk3 protein, A. thaliana), SEQ ID NO: 106 (ORF of the Mapkkk5 protein, A. thaliana), SEQ ID NO: 107 (ORF of the Mfp2 protein, A. thaliana), SEQ ID NO: 108 (ORF of the Mrb1 protein, A. thaliana), SEQ ID NO: 109 (ORF of the Nsp1 protein, M. truncatula), SEQ ID NO: 110 (ORF of the Nsp1 protein, A. thaliana), SEQ ID NO: 111 (ORF of the Nsp1 protein, B. distachyon), SEQ ID NO: 112 (ORF of the Nsp1 protein, G. max), SEQ ID NO: 113 (ORF of the Nsp1 protein, G. max), SEQ ID NO: 114 (ORF of the Nsp1 protein, O. sativa), SEQ ID NO: 115 (ORF of the Nsp1 protein, S. lycopersicum), SEQ ID NO: 116 ( ORF of the Nsp1 protein, S. lycopersicum), SEQ ID NO: 117 (ORF of the Nsp1 protein, Z. mays), SEQ ID NO: 118 (ORF of the Nsp1 protein, Z. mays), SEQ ID NO: 119 (ORF of the Nsp1 protein, Z. mays), SEQ ID NO: 120 (ORF of the Nsp1 protein, Z. mays), SEQ ID NO: 121 (ORF of the Nsp1 protein, B. rapa), SEQ ID NO: 122 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 123 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 124 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO : 125 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 126 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 127 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 128 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 129 (ORF of the Nsp1 protein, S. tuberosum), SEQ ID NO: 130 (ORF of the Nsp1 protein, S. tuberosum), SEQ ID NO: 131 (ORF of the Nsp1 protein, T. aestivum), SEQ ID NO: 132 (ORF of the Nsp1 protein, T. aestivum), SEQ ID NO: 133 (ORF of the Nsp1 protein, L. perenne), SEQ ID NO: 134 (ORF of the Nsp1 protein, L. perenne), SEQ ID NO: 135 (ORF of the Pds protein, A. thaliana), SEQ ID NO: 136 (ORF of the Pen3 protein, A. thaliana) , SEQ ID NO: 137 (ORF of the Phyb protein, A. thaliana), SEQ ID NO: 138 (ORF of the Pif3 protein, A. thaliana), SEQ ID NO: 139 (ORF of the Pizza protein, A thaliana), SEQ ID NO: 140 (ORF of the Ppoxl protein, A thaliana), SEQ ID NO: 141 (ORF of the Ppox2 protein, A. thaliana), SEQ ID NO: 142 ( ORF of the Prp39 protein, A. thaliana), SEQ ID NO: 143 (ORF of the PsbA protein, A. thaliana), SEQ ID NO: 144 (ORF of the Pskrl protein, A. thaliana), SEQ ID NO: 145 (ORF of the Rd21 protein, A. thaliana), SEQ ID NO: 146 (ORF of the Ringl protein, A. thaliana), SEQ ID NO: 147 (ORF of the Rosi protein, A. thaliana), SEQ ID NO: 148 (ORF of the Rpt4a protein, A. thaliana), SEQ ID NO: 149 (ORF of the Sfr6 protein, A. thaliana), SEQ ID NO: 150 (ORF of the Shr protein, A. thaliana), SEQ ID NO : 151 (ORF of the Shy2 protein, A. thaliana), SEQ ID NO: 152 (ORF of the Ski protein, M. truncatula), SEQ ID NO: 153 (ORF of the Sps1 protein, A. thaliana), SEQ ID NO: 154 (ORF of the Spt protein, A. thaliana), SEQ ID NO: 155 (ORF of the Stn8 protein, A. thaliana), SEQ ID NO: 156 (ORF of the Tap46 protein, A. thaliana), SEQ ID NO: 157 (ORF of the Topp6 protein, A. thaliana), SEQ ID NO: 158 (ORF of the TubB6 protein, A. thaliana), SEQ ID NO: 159 (ORF of the TubB8 protein, A. thaliana), SEQ ID NO: 160 (ORF of the llbala protein, A. thaliana), SEQ ID NO: 161 (ORF of the Vim3 protein, A. thaliana), SEQ ID NO: 381 (ORF of the Sgr1 protein, A. thaliana) , SEQ ID NO: 382 (ORF of the Abi5 protein, A. thaliana), SEQ ID NO: 383 (ORF of the Hsp101 protein, A. thaliana), SEQ ID NO: 384 (ORF of the Rh10 protein, M. truncatula ) and SEQ ID NO: 385 (ORF of the Wus protein, A. thaliana).
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity. , preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1 ).
En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Par « pourcentage d’identité » entre deux séquences d’acides nucléiques (ou d’acides aminés), on entend un pourcentage de nucléotides (ou de résidus d’acides aminés) identiques entre les deux séquences à comparer, obtenu après le meilleur alignement. Ce pourcentage est purement statistique et les différences entre les deux séquences sont réparties au hasard sur toute la longueur des séquences. Le meilleur alignement (ou alignement optimal) est l’alignement pour lequel le pourcentage d’identité entre les deux séquences à comparer, comme calculé ci- après, est le plus élevé. Les comparaisons de séquences entre deux séquences d’acides nucléiques (ou d’acides aminés) sont traditionnellement réalisées en comparant ces séquences après les avoir alignées de manière optimale, ladite comparaison étant réalisée par segment ou par fenêtre de comparaison pour identifier et comparer les régions locales de similarité de séquence. L’alignement optimal des séquences pour la comparaison peut être réalisé manuellement ou au moyen d’algorithmes et de logiciels à la disposition de l’homme de l’art, par exemple, la plateforme BLAST ou le programme MatGat (Campanella, Bitincka and Smalley, 2003). By “percentage of identity” between two nucleic acid (or amino acid) sequences, we mean a percentage of nucleotides (or amino acid residues) identical between the two sequences to be compared, obtained after the best alignment. . This percentage is purely statistical and the differences between the two sequences are randomly distributed over the entire length of the sequences. The best alignment (or optimal alignment) is the alignment for which the percentage of identity between the two sequences to be compared, as calculated below, is the highest. Sequence comparisons between two nucleic acid (or amino acid) sequences are traditionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out per segment or per comparison window to identify and compare the regions local sequence similarity. The optimal alignment of sequences for comparison can be carried out manually or by means of algorithms and software available to those skilled in the art, for example, the BLAST platform or the MatGat program (Campanella, Bitincka and Smalley , 2003).
Le pourcentage d’identité entre deux séquences est déterminé en comparant ces deux séquences alignées de manière optimale par fenêtre de comparaison dans laquelle la région de la séquence à comparer peut comprendre des additions ou des délétions par rapport à la séquence de référence pour un alignement optimal entre ces deux séquences. Le pourcentage d’identité est calculé en déterminant le nombre de positions identiques pour lesquelles le nucléotide (ou l’acide aminé) est identique entre les deux séquences, en divisant ce nombre de positions identiques par le nombre total de positions dans la fenêtre de comparaison et en multipliant le résultat obtenu par 100. The percentage identity between two sequences is determined by comparing these two optimally aligned sequences by comparison window in which the region of the sequence to be compared may include additions or deletions relative to the reference sequence for optimal alignment between these two sequences. Percent identity is calculated by determining the number of identical positions for which the nucleotide (or amino acid) is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100.
Au sens de l’invention, il est entendu dans l’invention que des séquences présentant « au moins 80% d’identité » avec une séquence de référence peuvent notamment présenter au moins 80%, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % ou 100 % d’identité avec ladite séquence de référence. For the purposes of the invention, it is understood in the invention that sequences presenting “at least 80% identity” with a reference sequence may in particular present at least 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% d identity with said reference sequence.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. Dans un mode de réalisation, l’invention concerne également le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs : 1 to 161 and 381 to 385. In one embodiment, the invention also relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising a sequence of nucleic acids chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un autre mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est choisie parmi les séquences : SEQ ID NO : 162 (cPEPcpk3), SEQ ID NO : 163 (cPEPdcll), SEQ ID NO : 164 (cPEPnsp1_3), SEQ ID NO : 165 (cPEPnsp1_1), SEQ ID NO : 166 (cPEPnsp1_2), SEQ ID NO : 167 (cPEPnsp1_4), SEQ ID NO : 168 (cPEPnsp1_5), SEQ ID NO : 169 (cPEPnspl 5aa), SEQ ID NO : 170 (cPEPnspl 20aa), SEQ ID NO : 171 (cPEPnspl 30aa), SEQ ID NO : 172 (cPEPnspl 40aa), SEQ ID NO : 173 (cPEPnspl 60aa), SEQ ID NO : 174 (cPEPnspl 80aa), SEQ ID NO : 175 (cPEPgapc), SEQ ID NO : 176 (cPEPbril), SEQ ID NO : 177 (cPEPbakl), SEQ ID NO : 178 (cPEPshy2), SEQ ID NO : 179 (cPEPpizza), SEQ ID NO : 180 (cPEPmrbl), SEQ ID NO : 181 (cPEPtap46), SEQ ID NO : 182 (cPEPspt), SEQ ID NO : 183 (cPEPga2ox7), SEQ ID NO : 184 (cPEPphyb), SEQ ID NO : 185 (cPEPhagô), SEQ ID NO : 186 (cPEPshr), SEQ ID NO : 187 (cPEPmapkkk3), SEQ ID NO : 188 (cPEPmapkkk5_1), SEQ ID NO : 189 (cPEPmapkkk5_2), SEQ ID NO : 190 (cPEPringl), SEQ ID NO : 191 (cPEProsI), SEQ ID NO : 192 (cPEPjarl), SEQ ID NO : 193 (cPEPcoil), SEQ ID NO : 194 (cPEPabcg34), SEQ ID NO : 195 (cPEPagbl), SEQ ID NO : 196 (cPEPwus), SEQ ID NO : 197 (AhEIN2), SEQ ID NO : 198 (AhBRH), SEQ ID NO : 199 (AhBAKI), SEQ ID NO : 200 (AtEIN2cPEP1), SEQ ID NO : 201 (AtEIN2cPEP2), SEQ ID NO : 202 (AtEIN2cPEP3), SEQ ID NO : 203 (AtEIN2cPEP13), SEQ ID NO : 204 (cPEPein2_1), SEQ ID NO : 205 (cPEPein2_2), SEQ ID NO : 206 (cPEPein2_3), SEQ ID NO : 404 (cPEPhsp101), SEQ ID NO : 406 (cPEPabiô), SEQ ID NO : 407 (cPEPsgrl), SEQ ID NO : 408 (cPEPhsp101), SEQ ID NO : 409 (cPEPmrbl), SEQ ID NO : 410 (cPEPshy2), SEQ ID NO : 411 (cPEPskl), SEQ ID NO : 412 (cPEPrhIO), SEQ ID NO : 413 (cPEpjarl), SEQ ID NO : 414 (cPEPbakl), SEQ ID NO : 415 (cPEPbril), SEQ ID NO : 416 (cPEPwus) et SEQ ID NO : 417 (cPEPein2). En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est choisie parmi les séquences : SEQ ID NO : 162, SEQ ID NO : 163 et SEQ ID NOs : 164 à 174. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est la séquence SEQ ID NO : 162. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est la séquence : SEQ ID NO : 163. En particulier, l’invention concerne le procédé de préparation et de détermination d’un cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est choisie parmi les séquences : SEQ ID NOs : 164 à 174. In another embodiment, the invention relates to the method for preparing and determining a cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NO: 162 (cPEPcpk3), SEQ ID NO: 163 (cPEPdcll), SEQ ID NO: 164 (cPEPnsp1_3), SEQ ID NO: 165 (cPEPnsp1_1), SEQ ID NO: 166 (cPEPnsp1_2), SEQ ID NO: 167 (cPEPnsp1_4), SEQ ID NO: 168 (cPEPnsp1_5 ), SEQ ID NO: 169 (cPEPnspl 5aa), SEQ ID NO: 170 (cPEPnspl 20aa), SEQ ID NO: 171 (cPEPnspl 30aa), SEQ ID NO: 172 (cPEPnspl 40aa), SEQ ID NO: 173 (cPEPnspl 60aa ), SEQ ID NO: 174 (cPEPnspl 80aa), SEQ ID NO: 175 (cPEPgapc), SEQ ID NO: 176 (cPEPbril), SEQ ID NO: 177 (cPEPbakl), SEQ ID NO: 178 (cPEPshy2), SEQ ID NO: 179 (cPEPpizza), SEQ ID NO: 180 (cPEPmrbl), SEQ ID NO: 181 (cPEPtap46), SEQ ID NO: 182 (cPEPspt), SEQ ID NO: 183 (cPEPga2ox7), SEQ ID NO: 184 (cPEPphyb ), SEQ ID NO: 185 (cPEPhagô), SEQ ID NO: 186 (cPEPshr), SEQ ID NO: 187 (cPEPmapkkk3), SEQ ID NO: 188 (cPEPmapkkk5_1), SEQ ID NO: 189 (cPEPmapkkk5_2), SEQ ID NO : 190 (cPEPringl), SEQ ID NO: 191 (cPEProsI), SEQ ID NO: 192 (cPEPjarl), SEQ ID NO: 193 (cPEPcoil), SEQ ID NO: 194 (cPEPabcg34), SEQ ID NO: 195 (cPEPagbl) , SEQ ID NO: 196 (cPEPwus), SEQ ID NO: 197 (AhEIN2), SEQ ID NO: 198 (AhBRH), SEQ ID NO: 199 (AhBAKI), SEQ ID NO: 200 (AtEIN2cPEP1), SEQ ID NO: 201 (AtEIN2cPEP2), SEQ ID NO: 202 (AtEIN2cPEP3), SEQ ID NO: 203 (AtEIN2cPEP13), SEQ ID NO: 204 (cPEPein2_1), SEQ ID NO: 205 (cPEPein2_2), SEQ ID NO: 206 (cPEPein2_3), SEQ ID NO: 404 (cPEPhsp101), SEQ ID NO: 406 (cPEPabiô), SEQ ID NO: 407 (cPEPsgrl), SEQ ID NO: 408 (cPEPhsp101), SEQ ID NO: 409 (cPEPmrbl), SEQ ID NO: 410 (cPEPshy2), SEQ ID NO: 411 (cPEPskl), SEQ ID NO: 412 (cPEPrhIO), SEQ ID NO: 413 (cPEpjarl), SEQ ID NO: 414 (cPEPbakl), SEQ ID NO: 415 (cPEPbril), SEQ ID NO: 416 (cPEPwus) and SEQ ID NO: 417 (cPEPein2). In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NOs : 164 to 174. In particular, the invention relates to the method for preparing and determining a cPEP as described above, in which the sequence of said peptide is the sequence SEQ ID NO: 162. In particular, the invention relates the process for preparing and determining a cPEP as described above, in which the sequence of said peptide is the sequence: SEQ ID NO: 163. In particular, the invention relates to the process for preparing and determining a cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NOs: 164 to 174.
Dans un deuxième aspect, l’invention ci-dessus a pour objet un cPEP tel qu’obtenu par la mise en œuvre du procédé tel que décrit précédemment. Selon ce même aspect, l’invention a également pour objet un cPEP, de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment (non naturellement traduit) d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In a second aspect, the subject of the invention above is a cPEP as obtained by implementing the method as described above. According to this same aspect, the invention also relates to a cPEP, of 4 to 70 amino acids, in particular of 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment (not naturally translated) of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne le cPEP tel que précédemment décrit, ledit fragment ayant une taille de 3n nucléotides, n étant compris : de 4 à 41 ; de 5 à 40 ; de 7 à 20 ; ou de 8 à 15. In one embodiment, the invention relates to cPEP as previously described, said fragment having a size of 3n nucleotides, n being included: from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
Autrement dit, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP comprenant s, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 ou 70 acides aminés. In other words, the invention relates to cPEP as described above, said cPEP comprising s, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids .
En particulier, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP comprenant de 4 à 41 acides aminés. En particulier, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP comprenant de 5 à 40 acides aminés. En particulier, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP comprenant de 7 à 20 acides aminés. En particulier, l’invention concerne également le cPEP tel que décrit précédemment, ledit cPEP comprenant de 8 à 15 acides aminés. In particular, the invention relates to cPEP as described above, said cPEP comprising from 4 to 41 amino acids. In particular, the invention relates to cPEP as described above, said cPEP comprising from 5 to 40 amino acids. In particular, the invention relates to cPEP as described above, said cPEP comprising from 7 to 20 amino acids. In particular, the invention also relates to cPEP as described above, said cPEP comprising 8 to 15 amino acids.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la taille dudit cPEP est inférieure à celle de ladite protéine. In one embodiment, the invention relates to cPEP as described above, in which the size of said cPEP is less than that of said protein.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, ledit fragment étant dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to cPEP as described above, said fragment lacking: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne donc le cPEP tel que décrit précédemment, ledit fragment comprenant un codon initiateur AUG codant une méthionine initiatrice et étant dépourvu d’un codon STOP choisi parmi les codons : UAG, UGA et UAA. L’invention concerne également le cPEP tel que décrit précédemment, ledit fragment étant dépourvu d’un codon initiateur AUG codant une méthionine initiatrice et comprenant un codon STOP choisi parmi les codons : UAG, UGA et UAA. In one embodiment, the invention therefore relates to cPEP as described above, said fragment comprising an AUG initiator codon encoding an initiator methionine and being devoid of a STOP codon chosen from the codons: UAG, UGA and UAA. The invention also relates to cPEP as described above, said fragment being devoid of an AUG initiator codon encoding an initiator methionine and comprising a STOP codon chosen from the codons: UAG, UGA and UAA.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, ledit fragment étant dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; et d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et dans lequel ledit fragment est choisi : soit dans le même cadre de lecture que le cadre ouvert de lecture codant ladite protéine ; soit dans un cadre de lecture décalé d’un ou deux nucléotides par rapport au cadre ouvert de lecture codant ladite protéine. In one embodiment, the invention relates to cPEP as described above, said fragment lacking: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and in which said fragment is chosen: either in the same reading frame as the open reading frame encoding said protein; either in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans le même cadre de lecture que le cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans le cadre de lecture déterminé par le codon d’initiation du cadre ouvert de lecture de ladite protéine. In one embodiment, the invention relates to cPEP as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the naturally translated nucleic acid sequence. in said plant cell. In other words, the invention relates to cPEP as described previously, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également le cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention relates to cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or two nucleotides in 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention a pour objet un cPEP, de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques d’un ARNm d’une protéine, ladite séquence d’acides nucléiques comprenant deux parties contiguës : une partie située au sein d’une séquence d’acides nucléiques réputée non codante (/.e. non traduite naturellement, e.g. 3’IITR ou 5’IITR) ; et une partie située au sein d’une séquence d’acides nucléiques réputée codante (/.e. traduite naturellement, e.g. exon), ledit fragment ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans une cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In one embodiment, the subject of the invention is a cPEP, of 4 to 70 amino acids, in particular of 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence of an mRNA of a protein, said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/e. non translated naturally, e.g. 3'IITR or 5'IITR); and a part located within a nucleic acid sequence deemed to be coding (/e. naturally translated, e.g. exon), said fragment having a size of 3n nucleotides, n being included from 4 to 70, in particular n being included from 4 to 41, and said cPEP being capable of modulating the accumulation of said protein in a plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ledit cPEP est capable d’augmenter l’accumulation de ladite protéine dans ladite cellule végétale. Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ledit cPEP est capable diminuer l’accumulation de ladite protéine dans ladite cellule végétale. In one embodiment, the invention relates to cPEP as described above, wherein said cPEP is capable of increasing the accumulation of said protein in said plant cell. In one embodiment, the invention relates to cPEP as described above, in which said cPEP is capable of reducing the accumulation of said protein in said plant cell.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ledit cPEP est un peptide synthétique. In one embodiment, the invention relates to cPEP as described above, in which said cPEP is a synthetic peptide.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ledit cPEP est un peptide isolé. In one embodiment, the invention relates to cPEP as described above, in which said cPEP is an isolated peptide.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ledit cPEP est un peptide recombinant. In one embodiment, the invention relates to cPEP as described above, in which said cPEP is a recombinant peptide.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP étant un peptide hydrophobe ou un peptide hydrophile. In one embodiment, the invention relates to cPEP as described above, said cPEP being a hydrophobic peptide or a hydrophilic peptide.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est naturellement présente dans ladite cellule végétale. In one embodiment, the invention relates to cPEP as described above, in which said protein is naturally present in said plant cell.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine n’est pas présente naturellement dans ladite cellule végétale. En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un transgène introduit artificiellement dans ladite cellule végétale. En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un vecteur introduit artificiellement dans ladite cellule végétale. In one embodiment, the invention relates to cPEP as described above, in which said protein is not naturally present in said plant cell. In particular, the invention relates to cPEP as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell. In particular, the invention relates to cPEP as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite cellule végétale (/.e. celle dans laquelle on souhaite moduler l’accumulation d’une protéine) appartient à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite cellule végétale est une cellule d’une algue. In one embodiment, the invention relates to cPEP as described above, in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn). In one embodiment, the invention relates to cPEP as described above, in which said plant cell is a cell of an alga.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi : Aae15, Aae16, abcg11, Abdcg34, Acd, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, RhIO et Wus. In one embodiment, the invention relates to cPEP as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acd, Agb1, Als, Anac076, Apg9, Arlbl, Arr1 , Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9 , Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1 , Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, RhIO and Wus.
En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi les gènes : Cpk3, Dell et Nsp1. En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Cpk3. En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Dell. En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Nsp1. In particular, the invention relates to cPEP as described above, in which said protein is encoded by a gene chosen from the genes: Cpk3, Dell and Nsp1. In particular, the invention relates to cPEP as described above, in which said protein is encoded by the Cpk3 gene. In particular, the invention relates to cPEP as described above, in which said protein is encoded by the Dell gene. In particular, the invention relates to cPEP as described above, in which said protein is encoded by the Nsp1 gene.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. In one embodiment, the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
En particulier, l’invention concerne l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). En particulier, l’invention concerne également le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne également le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne également le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1). In particular, the invention also relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention also relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In in particular, the invention also relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. L’invention concerne notamment le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385. The invention relates in particular to cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un autre mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est choisie parmi les séquences : SEQ ID NOs : 162 à 206, 404 et 406 à 417. In another embodiment, the invention relates to cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NOs: 162 to 206, 404 and 406 to 417.
En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est choisie parmi les séquences : SEQ ID NO : 162, SEQ ID NO : 163 et SEQ ID NOs : 164 à 174. En particulier, l’invention le cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est la séquence SEQ ID NO : 162. En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est la séquence : SEQ ID NO : 163. En particulier, l’invention concerne le cPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est choisie parmi les séquences : SEQ ID NOs : 164 à 174. In particular, the invention relates to cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NOs: 164 to 174. In particular, the invention the cPEP as described previously, in which the sequence of said peptide is the sequence SEQ ID NO: 162. In particular, the invention relates to the cPEP as described previously, in which the sequence of said peptide is the sequence: SEQ ID NO: 163. In particular, the invention relates to cPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NOs: 164 to 174.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, dans lequel ladite protéine est impliquée dans au moins un phénotype de plante choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In one embodiment, the invention relates to cPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Dans l’invention, un cPEP peut être fusionné ou lié à une ou plusieurs molécules facilitant l’entrée du cPEP dans la cellule. Parmi ces molécules, on peut notamment citer des peptides pénétrants (Numata, K., et al. Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Sci Rep 8, 10966 (2018).) et l’acide palmitique. Par « peptide pénétrant » (ci-après CPP), on entend des peptides de petite taille pénétrant les bicouches lipidiques cellulaires ou déstabilisent les membranes cellulaires. Les CPP peuvent être classés en trois groupes : cationiques, amphipathiques et hydrophobes. En particulier : les CPP cationiques contiennent de nombreux acides aminés chargés positivement, tels que la lysine (Lys) et l'arginine (Arg) ; les CPP amphipathiques sont généralement composés d'une séquence alternée d'acides aminés polaires et non polaires ; et les CPP hydrophobes se composent d'acides aminés non polaires avec des charges nettes relativement faibles. In the invention, a cPEP may be fused or linked to one or more molecules facilitating entry of the cPEP into the cell. Among these molecules, we can in particular cite penetrating peptides (Numata, K., et al. Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Sci Rep 8 , 10966 (2018).) and palmitic acid. By “penetrating peptide” (hereinafter CPP), we mean small peptides penetrating cellular lipid bilayers or destabilizing cell membranes. CPPs can be classified into three groups: cationic, amphipathic and hydrophobic. In particular: cationic CPPs contain many positively charged amino acids, such as lysine (Lys) and arginine (Arg); amphipathic CPPs are generally composed of an alternating sequence of polar and nonpolar amino acids; and hydrophobic CPPs consist of nonpolar amino acids with relatively low net charges.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP étant fusionné à un peptide facilitant son entrée dans la cellule végétale. En particulier, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP étant fusionné à un peptide pénétrant. In one embodiment, the invention relates to cPEP as described above, said cPEP being fused to a peptide facilitating its entry into the plant cell. In particular, the invention relates to cPEP as described above, said cPEP being fused to a penetrating peptide.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP étant fusionné à l’extrémité N-terminale ou à l’extrémité C-terminale avec ledit peptide facilitant son entrée dans la cellule végétale. En particulier, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP étant fusionné à l’extrémité N-terminale ou à l’extrémité C-terminale avec ledit peptide pénétrant. Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP étant fusionné avec : le peptide TAT (SEQ ID NO : 380) ; la pénétratine ; un peptide polyhistidine (notamment un peptide d’au moins 4 résidus histidine) ; ou un peptide polyarginine (notamment un peptide de 4 résidus arginine). In one embodiment, the invention relates to cPEP as described above, said cPEP being fused at the N-terminal end or at the C-terminal end with said peptide facilitating its entry into the plant cell. In particular, the invention relates to cPEP as described above, said cPEP being fused at the N-terminus or at the C-terminus with said penetrating peptide. In one embodiment, the invention relates to cPEP as described above, said cPEP being fused with: the TAT peptide (SEQ ID NO: 380); penetratin; a polyhistidine peptide (in particular a peptide of at least 4 histidine residues); or a polyarginine peptide (in particular a peptide of 4 arginine residues).
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP étant lié à une ou plusieurs molécules d’acide palmitique. In one embodiment, the invention relates to cPEP as described above, said cPEP being linked to one or more palmitic acid molecules.
Dans un mode de réalisation, l’invention concerne le cPEP tel que décrit précédemment, ledit cPEP étant lié à l’extrémité N-terminale ou à l’extrémité C-terminale à une ou plusieurs molécules d’acide palmitique. In one embodiment, the invention relates to cPEP as described above, said cPEP being linked at the N-terminus or at the C-terminus to one or more palmitic acid molecules.
Sur ce point, il convient de noter que la quantité de cPEP nécessaire pour moduler l’accumulation d’une protéine peut varier selon que le cPEP soit ou non modifié avec l’une des molécules facilitant sa pénétration cellulaire. On this point, it should be noted that the quantity of cPEP necessary to modulate the accumulation of a protein can vary depending on whether or not the cPEP is modified with one of the molecules facilitating its cellular penetration.
Dans un troisième aspect, l’invention ci-dessus a pour objet un acide nucléique codant un cPEP tel que décrit précédemment. Selon ce même aspect, l’invention a également pour objet un acide nucléique de 3n nucléotides, lequel acide nucléique correspond à un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm. In a third aspect, the invention above relates to a nucleic acid encoding a cPEP as described above. According to this same aspect, the invention also relates to a nucleic acid of 3n nucleotides, which nucleic acid corresponds to a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA.
Dans un mode de réalisation, l’invention concerne l’acide nucléique que décrit précédemment, ledit fragment étant dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the nucleic acid described above, said fragment lacking: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne donc l’acide nucléique tel que décrit précédemment, ledit fragment comprenant un codon initiateur AUG codant une méthionine initiatrice et étant dépourvu d’un codon STOP choisi parmi les codons : UAG, UGA et UAA. L’invention concerne également l’acide nucléique tel que décrit précédemment, ledit fragment étant dépourvu d’un codon initiateur AUG codant une méthionine initiatrice et comprenant un codon STOP choisi parmi les codons : UAG, UGA et UAA. In one embodiment, the invention therefore relates to the nucleic acid as described above, said fragment comprising an AUG initiator codon encoding an initiator methionine and being devoid of a STOP codon chosen from the codons: UAG, UGA and UAA. The invention also relates to the nucleic acid as described above, said fragment being devoid of an AUG initiator codon encoding an initiator methionine and comprising a STOP codon chosen from the codons: UAG, UGA and UAA.
Dans un mode de réalisation, l’invention concerne l’acide nucléique que décrit précédemment, ledit fragment étant dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; et d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the nucleic acid described above, said fragment lacking: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans le même cadre de lecture que le cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans le cadre de lecture déterminé par le codon d’initiation du cadre ouvert de lecture de ladite protéine. In one embodiment, the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the nucleic acid sequence. naturally translated in said plant cell. In other words, the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
Dans un mode de réalisation, l’invention concerne l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Dans un mode de réalisation, l’invention concerne un acide nucléique de 3n nucléotides, lequel acide nucléique correspond à un fragment d’une séquence d’acides nucléiques d’un ARNm d’une protéine, ladite séquence d’acides nucléiques comprenant deux parties contiguës : une partie située au sein d’une séquence d’acides nucléiques réputée non codante (/.e. non traduite naturellement, e.g. 3’UTR ou 5’IITR) ; et une partie située au sein d’une séquence d’acides nucléiques réputée codante (/.e. traduite naturellement, e.g. exon). In one embodiment, the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally occurring nucleic acid sequence. translated into said plant cell. In particular, the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in the 3' (or by two nucleotides in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5' ) relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3 ' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In one embodiment, the invention relates to a nucleic acid of 3n nucleotides, which nucleic acid corresponds to a fragment of a nucleic acid sequence of an mRNA of a protein, said nucleic acid sequence comprising two parts contiguous: a part located within a nucleic acid sequence deemed non-coding (/e. not naturally translated, eg 3'UTR or 5'IITR); and a part located within a nucleic acid sequence known to be coding (/e. naturally translated, eg exon).
En particulier, l’invention concerne l’acide nucléique tel que décrit précédemment, où n est compris : de 4 à 70 ; de 4 à 41 ; de 5 à 40 ; de 7 à 20 ; ou de 8 à 15. In particular, the invention relates to the nucleic acid as described above, where n is comprised: from 4 to 70; from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
Dans un autre aspect, l’invention ci-dessus concerne une composition comprenant un cPEP tel que décrit ci-dessus en tant que substance active. In another aspect, the above invention relates to a composition comprising a cPEP as described above as an active substance.
Dans un mode de réalisation, l’invention concerne une composition comprenant un cPEP en tant que substance active, ledit cPEP : In one embodiment, the invention relates to a composition comprising a cPEP as active substance, said cPEP:
- ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm ; et - having a size of 4 to 70 amino acids, in particular 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA; And
- étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. - being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the composition as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne donc la composition telle que décrite précédemment, dans laquelle ledit fragment comprend un codon initiateur AUG codant une méthionine initiatrice et est dépourvu d’un codon STOP choisi parmi les codons : UAG, UGA et UAA. L’invention concerne également la composition telle que décrite précédemment, dans laquelle ledit fragment est dépourvu d’un codon initiateur AUG codant une méthionine initiatrice et comprend un codon STOP choisi parmi les codons : UAG, UGA et UAA. In one embodiment, the invention therefore relates to the composition as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA. The invention also relates to the composition as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; et d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the composition as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans le même cadre de lecture que le cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans le cadre de lecture déterminé par le codon d’initiation du cadre ouvert de lecture de ladite protéine. In one embodiment, the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the naturally translated nucleic acid sequence. in said plant cell. In other words, the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relation to the open reading frame of the acid sequence nucleic acids naturally translated in said plant cell. In other words, the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne une composition comprenant un cPEP en tant que substance active, ledit cPEP : In one embodiment, the invention relates to a composition comprising a cPEP as active substance, said cPEP:
- ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit fragment ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ladite séquence d’acides nucléiques comprenant deux parties contiguës : - having a size of 4 to 70 amino acids, in particular 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being between 4 and 70, in particular n being between 4 and 41, and said nucleic acid sequence comprising two contiguous parts:
■ une partie située au sein d’une séquence d’acides nucléiques réputée non codante (/.e. non traduite naturellement, e.g. 3’IITR ou 5’IITR) ; et ■ a part located within a nucleic acid sequence deemed non-coding (/e. not naturally translated, e.g. 3’IITR or 5’IITR); And
■ une partie située au sein d’une séquence d’acides nucléiques réputée codante (/.e. traduite naturellement, e.g. exon) ; et ■ a part located within a nucleic acid sequence known to be coding (/e. naturally translated, e.g. exon); And
- étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. - being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit cPEP est à une concentration comprise de 10'9 M à 10'3 M. Sur ce point, il convient de noter d’une part que la composition de l’invention n’existe pas à l’état naturel et ceci est d’autant plus vrai qu’une telle concentration en cPEP ne peut exister au sein d’une cellule végétale. De plus et par « concentration comprise de 10'9 M à 10'3 M », on entend que la concentration en cPEP peut être comprise de 10'9 à 10'4 M, de 10'8 à 10'4 M, de 10'9 à 10'5 M, de 10'8 à 10'5 M, comme elle peut être comprise de 5 pM à 500 pM, de 30 pM à 70 pM, ou encore être de 50 pM. In one embodiment, the invention relates to the composition as described above, in which said cPEP is at a concentration comprised from 10'9 M to 10'3 M. On this point, it should be noted on the one hand that the composition of the invention does not exist in the natural state and this is all the more true since such a concentration of cPEP cannot exist within a plant cell. In addition and by "concentration comprised from 10' 9 M to 10' 3 M", it is understood that the concentration of cPEP can be comprised from 10' 9 to 10' 4 M, from 10' 8 to 10' 4 M, from 10' 9 to 10' 5 M, from 10' 8 to 10' 5 M, as it can be comprised from 5 pM to 500 pM, from 30 pM to 70 pM, or even be 50 pM.
En particulier, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit cPEP est à une concentration comprise de 10'9 à 10'4 M, de 10'8 à 10'4 M, de 10' 9 à 10'5 M ou de 10'8 à 10'5 M. En particulier, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit cPEP est à une concentration comprise de 5 pM à 500 pM ou de 30 pM à 70 pM. En particulier, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit cPEP est à une concentration de 50 pM. De manière non limitative, cette concentration peut également être de 10'9 M, 10'8 M, 10'7 M, 10'6 M, 10'5 M ou IO’4 M. In particular, the invention relates to the composition as described above, in which said cPEP is at a concentration comprised from 10' 9 to 10' 4 M, from 10' 8 to 10' 4 M, from 10' 9 to 10' 5 M or from 10'8 to 10'5 M. In particular, the invention relates to the composition as described above, in which said cPEP is at a concentration of from 5 pM to 500 pM or from 30 pM to 70 pM. In particular, the invention relates to the composition such as described previously, in which said cPEP is at a concentration of 50 pM. In a non-limiting manner, this concentration can also be 10' 9 M, 10' 8 M, 10' 7 M, 10' 6 M, 10' 5 M or IO' 4 M.
Au vu de ce qui précède, on comprend que l’invention concerne également la composition comprenant un cPEP en tant que substance active, ledit cPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm ; étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine ; et étant notamment à une concentration comprise de 5 pM à 500 pM ou de 30 pM à 70 pM, ou étant notamment à une concentration de 50 pM. In view of the above, it is understood that the invention also relates to the composition comprising a cPEP as active substance, said cPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, of which the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA ; being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein; and being in particular at a concentration of 5 pM to 500 pM or from 30 pM to 70 pM, or being in particular at a concentration of 50 pM.
A noter que par « composition comprenant un cPEP », on entend que la composition de l’invention comprend au moins un cPEP. C’est-à-dire qu’un mélange de cPEPs est envisageable, lesdits cPEPs pouvant cibler une même protéine ou plusieurs protéines selon le fragment d’acides nucléiques dont ils sont issus. A cet égard, les concentrations susmentionnées concernent soit le mélange de cPEPs en tant que tel, soit chacun des cPEPs dudit mélange, lesdits cPEPs pouvant être à la même concentration ou pouvant être à des concentrations différentes parmi celles citées ci-dessus. Note that by “composition comprising a cPEP”, we mean that the composition of the invention comprises at least one cPEP. That is to say, a mixture of cPEPs is possible, said cPEPs being able to target the same protein or several proteins depending on the nucleic acid fragment from which they come. In this regard, the aforementioned concentrations concern either the mixture of cPEPs as such, or each of the cPEPs of said mixture, said cPEPs being able to be at the same concentration or being able to be at different concentrations among those cited above.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition phytopharmaceutique, une composition herbicide ou une composition d’enrobage, en particulier ladite composition d’enrobage comprenant en outre au moins un agent de fixation. In one embodiment, the invention relates to the composition as described above, said composition being a phytopharmaceutical composition, a herbicidal composition or a coating composition, in particular said coating composition further comprising at least one fixing agent .
En particulier, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition phytopharmaceutique. En particulier, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition herbicide. En particulier, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition d’enrobage. De préférence, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition d’enrobage comprenant en outre au moins un agent de fixation. Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un solvant. De préférence, ledit solvant est choisi parmi : l’acétone, l’acétonitrile, l’acide acétique, l’acide formique, l’adipate de diméthyle, le benzyl acétate, le bi-butyl carbonate, le dimethyl sulfoxide (DMSO), l’eau, le glutarate de diméthyle, l’hydroxyde d’ammonium, l’iso-butanol, l’iso-propanol, le lactate de diéthyle héxyle, le solvant naphta aromatique léger, le solvant naphta aromatique lourd, le succinate de diéthyle et leurs mélanges (e.g. mélange [eau ; acide acétique] ; [acétonitrile ; acide acétique], [eau, acétonitrile ; acide acétique], [eau ; DMSO], [eau ; acétonitrile] ou [eau ; hydroxyde d’ammonium]). In particular, the invention relates to the composition as described above, said composition being a phytopharmaceutical composition. In particular, the invention relates to the composition as described above, said composition being a herbicidal composition. In particular, the invention relates to the composition as described above, said composition being a coating composition. Preferably, the invention relates to the composition as described above, said composition being a coating composition further comprising at least one fixing agent. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one solvent. Preferably, said solvent is chosen from: acetone, acetonitrile, acetic acid, formic acid, dimethyl adipate, benzyl acetate, bi-butyl carbonate, dimethyl sulfoxide (DMSO), water, dimethyl glutarate, ammonium hydroxide, iso-butanol, iso-propanol, diethyl hexyl lactate, light aromatic solvent naphtha, heavy aromatic solvent naphtha, diethyl succinate and their mixtures (eg mixture [water; acetic acid]; [acetonitrile; acetic acid], [water, acetonitrile; acetic acid], [water; DMSO], [water; acetonitrile] or [water; ammonium hydroxide]) .
Les propriétés de solubilité des cPEPs sont déterminées notamment par leur composition en acides aminés. Les cPEPs hydrophiles peuvent être solubilisés et conditionnés dans des solutions aqueuses, telles que l’eau. Les cPEPs hydrophobes peuvent être solubilisés et conditionnés dans des solvants, tels que les solvants organiques. The solubility properties of cPEPs are determined in particular by their amino acid composition. Hydrophilic cPEPs can be solubilized and packaged in aqueous solutions, such as water. Hydrophobic cPEPs can be solubilized and packaged in solvents, such as organic solvents.
Pour un traitement des plantes par les cPEPs, les solvants organiques sont des solvants non toxiques pour les plantes en faibles quantités, c’est-à-dire qu’ils n’ont pas d’effet délétère sur le développement de la plante. De manière non limitative, les solvants organiques peuvent être ceux cités ci-dessus et en particulier choisis parmi l’acétonitrile et l’acide acétique. For treatment of plants with cPEPs, organic solvents are non-toxic solvents for plants in small quantities, that is to say they have no deleterious effect on the development of the plant. In a non-limiting manner, the organic solvents may be those mentioned above and in particular chosen from acetonitrile and acetic acid.
Comme indiqué ci-dessus, les cPEPs peuvent également être solubilisés et conditionnés dans des mélanges de solvants, comme par exemple, un mélange de solvant organique [acétonitrile ; acide acétique], un mélange [eau ; DMSO] dans un ratio volume:volume compris de 99:1 à 1 :99, un mélange [eau ; acétonitrile] dans un ratio volume:volume compris de 99:1 à 1 :99 ou un mélange [eau ; hydroxyde d’ammonium] dans un ratio volume:volume compris de 99:1 à 99,9:0,1. Les cPEPs peuvent également être solubilisés dans une solution comprenant 50 % d’acétonitrile, 10 % d’acide acétique et 40 % d’eau (volume/volume/volume). As noted above, cPEPs can also be solubilized and packaged in solvent mixtures, such as, for example, an organic solvent mixture [acetonitrile; acetic acid], a mixture [water; DMSO] in a volume:volume ratio of 99:1 to 1:99, a mixture [water; acetonitrile] in a volume:volume ratio of 99:1 to 1:99 or a mixture [water; ammonium hydroxide] in a volume:volume ratio of 99:1 to 99.9:0.1. The cPEPs can also be solubilized in a solution comprising 50% acetonitrile, 10% acetic acid and 40% water (volume/volume/volume).
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un diluant. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one diluent.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un adjuvant. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one adjuvant.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un agent de fixation. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one fixing agent.
Par « agent de fixation », on entend un agent chimique ou naturel lequel permet de coller la composition de l’invention à une graine de végétal de manière à enrober ladite graine de végétal. On entend également une substance rendant possible l’application et la tenue de la ou des substances actives sur le grain. Parmi les agents de fixation disponibles, on retrouve notamment la carboxymethyl cellulose (CMC) et la gomme arabique. De plus et de manière non limitative, un agent de fixation peut comprendre des solvants organiques, de l’eau, des dispersants, des émulgateurs, des tensioactifs, des mouillants et des colorants. By “fixing agent” is meant a chemical or natural agent which makes it possible to stick the composition of the invention to a plant seed so as to coat said seed with vegetal. We also mean a substance making it possible to apply and hold the active substance(s) on the grain. Among the fixing agents available, we find in particular carboxymethyl cellulose (CMC) and gum arabic. Additionally and without limitation, a fixing agent may include organic solvents, water, dispersants, emulgators, surfactants, wetting agents and dyes.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un nutriment végétal. En particulier, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un agent de fixation et au moins un nutriment végétal. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one plant nutrient. In particular, the invention relates to the composition as described above, said composition further comprising at least one fixing agent and at least one plant nutrient.
Par « nutriment végétal », on entend un élément assimilé par la plante pour permettre son développement. De manière non limitative, un nutriment végétal peut être choisi parmi : l’azote, le phosphore, le potassium, le calcium, le magnésium, le soufre, le manganèse, le fer, le cuivre, le bore, le zinc, le molybdène et leurs mélanges. By “plant nutrient”, we mean an element assimilated by the plant to enable its development. In a non-limiting manner, a plant nutrient can be chosen from: nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, manganese, iron, copper, boron, zinc, molybdenum and their mixtures.
Au vu de ce qui précède, on comprend qu’un autre aspect de l’invention a pour objet une semence enrobée comprenant une graine de plante, ladite graine de plante étant enrobée par une composition d’enrobage telle que décrite précédemment. In view of the above, we understand that another aspect of the invention relates to a coated seed comprising a plant seed, said plant seed being coated with a coating composition as described above.
L’enrobage peut être réalisé selon les procédés classiquement utilisés dans l’industrie agroalimentaire et peut être obtenu en utilisant un matériau apte à se désagréger dans un solvant ou dans la terre, tel qu’un liant ou de l’argile. The coating can be carried out according to the processes conventionally used in the food industry and can be obtained by using a material capable of disintegrating in a solvent or in the earth, such as a binder or clay.
Selon l’invention, l’enrobage peut être utilisé pour conférer des propriétés particulières à une semence en combinaison avec un cPEP, telles qu’une croissance améliorée ou une résistance à certains stress biotiques ou abiotiques. According to the invention, the coating can be used to confer particular properties to a seed in combination with a cPEP, such as improved growth or resistance to certain biotic or abiotic stresses.
Dans un mode de réalisation, l’invention concerne la semence enrobée telle que décrite précédemment, dans lequel ladite graine de plante à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the coated seed as described above, in which said plant seed has a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (apple earth), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne la semence enrobée telle que décrite précédemment, ladite semence étant traitée par trempage dans une composition contenant un cPEP. Lors d’un trempage, la semence est alors plongée totalement ou partiellement dans une composition contenant un cPEP. In one embodiment, the invention relates to coated seed as described above, said seed being treated by soaking in a composition containing a cPEP. During soaking, the seed is then totally or partially immersed in a composition containing a cPEP.
Dans un autre aspect, l’invention ci-dessus concerne une utilisation d’un cPEP en tant qu’agent phytosanitaire pour moduler l’accumulation d’une protéine dans une cellule végétale, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In another aspect, the above invention relates to a use of a cPEP as a phytosanitary agent to modulate the accumulation of a protein in a plant cell, said cPEP having a size ranging from 4 to 70 amino acids , in particular from 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the framework reading open of said protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the use of a cPEP as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne donc l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit fragment comprend un codon initiateur AUG codant une méthionine initiatrice et est dépourvu d’un codon STOP choisi parmi les codons : UAG, UGA et UAA. L’invention concerne également l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit fragment est dépourvu d’un codon initiateur AUG codant une méthionine initiatrice et comprend un codon STOP choisi parmi les codons : UAG, UGA et UAA. Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; et d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention therefore relates to the use of a cPEP as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG , UGA and UAA. The invention also relates to the use of a cPEP as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA. In one embodiment, the invention relates to the use of a cPEP as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans le même cadre de lecture que le cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans le cadre de lecture déterminé par le codon d’initiation du cadre ouvert de lecture de ladite protéine. In one embodiment, the invention relates to the use of a cPEP as described previously, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the sequence d nucleic acids naturally translated in said plant cell. In other words, the invention relates to the use of a cPEP as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the use of a cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the sequence of nucleic acids naturally translated in said plant cell. In particular, the invention relates to the use of a cPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the use of a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in 3' (or by one nucleotide in 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the use of a cPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne une utilisation d’un cPEP en tant qu’agent phytosanitaire pour moduler l’accumulation d’une protéine dans une cellule végétale, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit fragment ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ladite séquence d’acides nucléiques comprenant deux parties contiguës : une partie située au sein d’une séquence d’acides nucléiques réputée non codante (/.e. non traduite naturellement, e.g. intron) ; et une partie située au sein d’une séquence d’acides nucléiques réputée codante (/.e. traduite naturellement, e.g. exon) ; ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In one embodiment, the invention relates to the use of a cPEP as a phytosanitary agent to modulate the accumulation of a protein in a plant cell, said cPEP having a size of 4 to 70 amino acids, in particular 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a naturally occurring nucleic acid sequence translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being between 4 and 70, in particular n being between 4 and 41, and said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/e. not naturally translated, eg intron); and a part located within a nucleic acid sequence known to be coding (/e. naturally translated, eg exon); said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment pour augmenter l’accumulation de ladite protéine dans la cellule végétale. La présence du cPEP fait que la quantité de ladite protéine dans la cellule végétale traitée est supérieure à celle dans une cellule végétale non traitée. In one embodiment, the invention relates to the use of a cPEP as described above to increase the accumulation of said protein in the plant cell. The presence of cPEP causes the quantity of said protein in the treated plant cell to be greater than that in an untreated plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment pour diminuer (inhiber) l’accumulation de ladite protéine dans la cellule végétale. La présence du cPEP fait que la quantité de ladite protéine dans la cellule végétale traitée est inférieure à celle dans une cellule végétale non traitée. In one embodiment, the invention relates to the use of a cPEP as described above to reduce (inhibit) the accumulation of said protein in the plant cell. The presence of cPEP causes the quantity of said protein in the treated plant cell to be lower than that in an untreated plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est produit hors de ladite cellule végétale avant d’être introduit dans ladite cellule végétale. In one embodiment, the invention relates to the use of a cPEP as described above, in which said cPEP is produced outside of said plant cell before being introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est un peptide synthétique. In one embodiment, the invention relates to the use of a cPEP as described above, in which said cPEP is a synthetic peptide.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est un peptide isolé. In one embodiment, the invention relates to the use of a cPEP as described above, in which said cPEP is an isolated peptide.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est un peptide recombinant. In one embodiment, the invention relates to the use of a cPEP as described above, in which said cPEP is a recombinant peptide.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est un peptide hydrophobe ou un peptide hydrophile. Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est introduit dans ladite cellule végétale sous la forme d’un acide nucléique codant ledit cPEP. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est introduit dans ladite cellule végétale sous la forme d’un acide nucléique codant ledit cPEP et comprenant les moyens de l’exprimer. In one embodiment, the invention relates to the use of a cPEP as described above, in which said cPEP is a hydrophobic peptide or a hydrophilic peptide. In one embodiment, the invention relates to the use of a cPEP as described above, in which said cPEP is introduced into said plant cell in the form of a nucleic acid encoding said cPEP. In particular, the invention relates to the use of a cPEP as described above, in which said cPEP is introduced into said plant cell in the form of a nucleic acid encoding said cPEP and comprising the means of expressing it.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est naturellement présente dans ladite cellule végétale. In one embodiment, the invention relates to the use of a cPEP as described above, in which said protein is naturally present in said plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine n’est pas présente naturellement dans ladite cellule végétale. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un transgène introduit artificiellement dans ladite cellule végétale. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un vecteur introduit artificiellement dans ladite cellule végétale. In one embodiment, the invention relates to the use of a cPEP as described above, in which said protein is not naturally present in said plant cell. In particular, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell. In particular, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle l’accumulation de ladite protéine est déterminée via la mise en œuvre d’une technique choisie parmi : le Western blot, la mesure de l’activité enzymatique, la spectrométrie de masse et la fusion traductionnelle. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle l’accumulation de ladite protéine est déterminée via la mise en œuvre d’un Western blot. In one embodiment, the invention relates to the use of a cPEP as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blotting, measurement of enzymatic activity, mass spectrometry and translational fusion. In particular, the invention relates to the use of a cPEP as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP a une taille comprise de 4 à 41 acides aminés, de 5 à 40 acides aminés, de 7 à 20 acides aminés ou plus particulièrement une taille comprise de 8 à 15 acides aminés. En particulier, ledit cPEP a une taille de 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,In one embodiment, the invention relates to the use of a cPEP as described above, in which said cPEP has a size comprised from 4 to 41 amino acids, from 5 to 40 amino acids, from 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids. In particular, said cPEP has a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69 ou 70 acides aminés. 64, 65, 66, 67, 68, 69 or 70 amino acids.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite cellule végétale appartient à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the use of a cPEP as described above, in which said plant cell belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii , Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite cellule végétale est une cellule d’une algue. In particular, the invention relates to the use of a cPEP as described above, in which said plant cell is a cell of an alga.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène choisi parmi : Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Aribl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 et Wus. In one embodiment, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Aribl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppo, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 and Wus.
Au vu de ce qui précède, on comprend que dans un autre mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment non naturellement traduit d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit fragment non naturellement traduit étant dépourvu du codon initiateur AUG codant une méthionine initiatrice et/ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et étant choisi soit dans le même cadre de lecture que le cadre ouvert de lecture codant ladite protéine, soit dans un cadre de lecture décalé d’un ou deux nucléotides par rapport au cadre ouvert de lecture codant ladite protéine, et ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, ladite protéine étant codée par un gène choisi parmi : Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubBô, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 et Wus. In view of the above, it is understood that in another embodiment, the invention relates to the use of a cPEP as described previously, said cPEP having a size of 4 to 70 amino acids, in particular 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a non-naturally translated fragment of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open frame of reading of said protein encoded by said mRNA, said non-naturally translated fragment being devoid of the AUG initiator codon encoding an initiator methionine and/or a STOP codon chosen from the codons: UAG, UGA and UAA, and being chosen either in the same reading frame that the open reading frame encoding said protein, is in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein, and said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein, said protein being encoded by a gene chosen from: Aae15, Aae16, abcg11 , Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26 , Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1 , Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubBô, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 and Wus.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. In one embodiment, the invention relates to the use of a cPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the use of a cPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. Dans un mode de réalisation, l’invention concerne également l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a sequence of nucleic acids chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385. In one embodiment, the invention also relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the use of a cPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un autre mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est choisi parmi les séquences : SEQ ID NOs : 162 à 206, 404 et 406 à 417. In another embodiment, the invention relates to the use of a cPEP as described above, in which said cPEP is chosen from the sequences: SEQ ID NOs: 162 to 206, 404 and 406 to 417.
En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est choisi parmi les séquences : SEQ ID NO : 162, SEQ ID NO : 163 et SEQ ID NOs : 164 à 174. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est de séquence SEQ ID NO : 162. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est de séquence : SEQ ID NO : 163. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est choisi parmi les séquences : SEQ ID NOs : 164 à 174. In particular, the invention relates to the use of a cPEP as described above, in which said cPEP is chosen from the sequences: SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NOs: 164 to 174. in particular, the invention relates to the use of a cPEP as described above, in which said cPEP is of sequence SEQ ID NO: 162. In particular, the invention relates to the use of a cPEP as described previously, in which said cPEP is of sequence: SEQ ID NO: 163. In particular, the invention relates to the use of a cPEP as described above, in which said cPEP is chosen from the sequences: SEQ ID NOs: 164 to 174 .
Dans un autre mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ladite protéine est impliquée dans au moins un phénotype de plante choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In another embodiment, the invention relates to the use of a cPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume , the mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Dans un autre mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, pour moduler l’accumulation d’une protéine recombinante dont la séquence d’acides nucléiques qui la code correspond à la fusion des séquences d’acides nucléiques de deux gènes distincts. In another embodiment, the invention relates to the use of a cPEP as described above, to modulate the accumulation of a recombinant protein whose nucleic acid sequence which encodes it corresponds to the fusion of the sequences of nucleic acids of two distinct genes.
En particulier, la séquence codante d’au moins un des deux gènes est celle d’un gène rapporteur, par exemple un gène codant une protéine fluorescente (telle que la GFP) ou une protéine permettant la résistance de la plante à un composé. In particular, the coding sequence of at least one of the two genes is that of a reporter gene, for example a gene encoding a fluorescent protein (such as GFP) or a protein allowing resistance of the plant to a compound.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, pour moduler l’accumulation d’une protéine recombinante dont la séquence d’acides nucléiques qui la code correspond à la fusion : d’une séquence d’acides nucléiques réputée non codante d’un premier gène ; et d’une séquence d’acides nucléiques codante d’un second gène, la séquence dudit cPEP correspondant à la traduction via le code génétique d’un fragment de la séquence d’acides nucléiques réputée non codante du premier gène. In one embodiment, the invention relates to the use of a cPEP as described above, to modulate the accumulation of a recombinant protein whose nucleic acid sequence which encodes it corresponds to the fusion: of a nucleic acid sequence deemed non-coding of a first gene; and a coding nucleic acid sequence of a second gene, the sequence of said cPEP corresponding to the translation via the genetic code of a fragment of the nucleic acid sequence deemed non-coding of the first gene.
Dans un autre aspect, l’invention ci-dessus concerne un procédé de modulation de l’accumulation d’une protéine dans une cellule végétale comprenant une étape d’introduction : d’un cPEP ; ou d’un acide nucléique codant ledit cPEP et les moyens de l’exprimer, dans ladite cellule végétale, l’introduction dudit cPEP entraînant une modulation de la quantité de ladite protéine dans ladite cellule végétale, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In another aspect, the above invention relates to a method of modulating the accumulation of a protein in a plant cell comprising a step of introducing: a cPEP; or a nucleic acid encoding said cPEP and the means of expressing it, in said plant cell, the introduction of said cPEP leading to a modulation of the quantity of said protein in said plant cell, said cPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit fragment étant dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the method as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne donc le procédé tel que décrit précédemment, dans lequel ledit fragment comprend un codon initiateur AUG codant une méthionine initiatrice et est dépourvu d’un codon STOP choisi parmi les codons : UAG, UGA et UAA. L’invention concerne également le procédé tel que décrit précédemment, dans lequel ledit fragment est dépourvu d’un codon initiateur AUG codant une méthionine initiatrice et comprend un codon STOP choisi parmi les codons : UAG, UGA et UAA. In one embodiment, the invention therefore relates to the method as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA. The invention also relates to the method as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit fragment étant dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; et d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the method as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans le même cadre de lecture que le cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans le cadre de lecture déterminé par le codon d’initiation du cadre ouvert de lecture de ladite protéine. In one embodiment, the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the naturally translated nucleic acid sequence. in said plant cell. In other words, the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a different reading frame. of the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or two nucleotides in 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne un procédé de modulation de l’accumulation d’une protéine dans une cellule végétale comprenant une étape d’introduction : d’un cPEP ; ou d’un acide nucléique codant ledit cPEP et les moyens de l’exprimer, dans ladite cellule végétale, l’introduction dudit cPEP entraînant une modulation de la quantité de ladite protéine dans ladite cellule végétale, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit fragment ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ladite séquence d’acides nucléiques comprenant deux parties contiguës : une partie située au sein d’une séquence d’acides nucléiques réputée non codante (/.e. non traduite naturellement, e.g. 3’IITR ou 5’IITR) ; et une partie située au sein d’une séquence d’acides nucléiques réputée codante (/.e. traduite naturellement, e.g. exon) ; ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In one embodiment, the invention relates to a method for modulating the accumulation of a protein in a plant cell comprising a step of introducing: a cPEP; or a nucleic acid encoding said cPEP and the means of expressing it, in said plant cell, the introduction of said cPEP leading to a modulation of the quantity of said protein in said plant cell, said cPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, and said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/e. not naturally translated, e.g. 3'IITR or 5'IITR); and a part located within a nucleic acid sequence known to be coding (/e. naturally translated, e.g. exon); said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, ledit procédé permettant : de favoriser le développement d’une plante ; ou de ralentir ou d’empêcher le développement d’une plante. In one embodiment, the invention relates to the method as described above, said method allowing: to promote the development of a plant; or slow down or prevent the development of a plant.
En particulier, l’invention concerne le procédé tel que décrit précédemment, ledit procédé permettant de favoriser le développement d’une plante. En particulier, l’invention concerne le procédé tel que décrit précédemment, ledit procédé permettant de ralentir ou d’empêcher le développement d’une plante. In particular, the invention relates to the process as described above, said process making it possible to promote the development of a plant. In particular, the invention relates to the process as described above, said process making it possible to slow down or prevent the development of a plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment pour augmenter l’accumulation de ladite protéine dans la cellule végétale. La présence du cPEP fait que la quantité de ladite protéine dans la cellule végétale traitée est supérieure à celle dans une cellule végétale non traitée. In one embodiment, the invention relates to the method as described above for increasing the accumulation of said protein in the plant cell. The presence of cPEP causes the quantity of said protein in the treated plant cell to be greater than that in an untreated plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment pour diminuer (inhiber) l’accumulation de ladite protéine dans la cellule végétale. La présence du cPEP fait que la quantité de ladite protéine dans la cellule végétale traitée est inférieure à celle dans une cellule végétale non traitée. In one embodiment, the invention relates to the method as described above for reducing (inhibiting) the accumulation of said protein in the plant cell. The presence of cPEP causes the quantity of said protein in the treated plant cell to be lower than that in an untreated plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est produit hors de ladite cellule végétale avant d’être introduit dans ladite cellule végétale. In one embodiment, the invention relates to the method as described above, in which said cPEP is produced outside of said plant cell before being introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est un peptide synthétique. In one embodiment, the invention relates to the method as described above, in which said cPEP is a synthetic peptide.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est un peptide isolé. In one embodiment, the invention relates to the method as described above, in which said cPEP is an isolated peptide.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est un peptide recombinant. In one embodiment, the invention relates to the method as described above, in which said cPEP is a recombinant peptide.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP étant un peptide hydrophobe ou un peptide hydrophile. In one embodiment, the invention relates to the method as described above, in which said cPEP being a hydrophobic peptide or a hydrophilic peptide.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est introduit dans ladite cellule végétale sous la forme d’un acide nucléique codant ledit cPEP. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est introduit dans ladite cellule végétale sous la forme d’un acide nucléique codant ledit cPEP et comprenant les moyens de l’exprimer. Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est naturellement présente dans ladite cellule végétale. In one embodiment, the invention relates to the method as described above, in which said cPEP is introduced into said plant cell in the form of a nucleic acid encoding said cPEP. In particular, the invention relates to the method as described above, in which said cPEP is introduced into said plant cell in the form of a nucleic acid encoding said cPEP and comprising the means of expressing it. In one embodiment, the invention relates to the method as described above, in which said protein is naturally present in said plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine n’est pas présente naturellement dans ladite cellule végétale. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un transgène introduit artificiellement dans ladite cellule végétale. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un vecteur introduit artificiellement dans ladite cellule végétale. In one embodiment, the invention relates to the method as described above, in which said protein is not naturally present in said plant cell. In particular, the invention relates to the method as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell. In particular, the invention relates to the method as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’accumulation de ladite protéine est déterminée via la mise en œuvre d’une technique choisie parmi : le Western blot, la mesure de l’activité enzymatique, la spectrométrie de masse et la fusion traductionnelle. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’accumulation de ladite protéine est déterminée via la mise en œuvre d’un Western blot. In one embodiment, the invention relates to the method as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blotting, measurement of enzymatic activity , mass spectrometry and translational fusion. In particular, the invention relates to the method as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP a une taille comprise de 4 à 41 acides aminés, de 5 à 40 acides aminés, de 7 à 20 acides aminés ou plus particulièrement une taille comprise de 8 à 15 acides aminés. En particulier, ledit cPEP a une taille de 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 ou 70 acides aminés. In one embodiment, the invention relates to the method as described above, in which said cPEP has a size comprised from 4 to 41 amino acids, from 5 to 40 amino acids, from 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids. In particular, said cPEP has a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite cellule végétale ou ladite plante appartient à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the method as described above, in which said plant cell or said plant belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata , Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean ), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite cellule végétale est une cellule d’une algue. In particular, the invention relates to the method as described above, in which said plant cell is an algae cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi : Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arrô, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, RhIO et Wus. In one embodiment, the invention relates to the method as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1 , Arr5, Arrô, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9 , Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1 , Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, RhIO and Wus.
Au vu de ce qui précède, on comprend que dans un autre mode de réalisation, l’invention concerne le procédé tel que décrit précédemment comprenant une étape d’introduction : d’un cPEP ; ou d’un acide nucléique codant ledit cPEP et les moyens de l’exprimer, dans ladite cellule végétale, l’introduction dudit cPEP entraînant une modulation de la quantité de ladite protéine dans ladite cellule végétale, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment non naturellement traduit d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit fragment non naturellement traduit étant dépourvu du codon initiateur AUG codant une méthionine initiatrice et/ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et étant choisi soit dans le même cadre de lecture que le cadre ouvert de lecture codant ladite protéine, soit dans un cadre de lecture décalé d’un ou deux nucléotides par rapport au cadre ouvert de lecture codant ladite protéine, et ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, ladite protéine étant codée par un gène choisi parmi : Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arrô, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Del 1, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gape, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Topp6, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 et Wus. In view of the above, it is understood that in another embodiment, the invention relates to the method as described above comprising a step of introducing: a cPEP; or a nucleic acid encoding said cPEP and the means of expressing it, in said plant cell, the introduction of said cPEP leading to a modulation of the quantity of said protein in said plant cell, said cPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a non-naturally translated fragment of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said non-naturally translated fragment being devoid of the AUG initiator codon encoding an initiator methionine and/or a STOP codon chosen from the codons: UAG, UGA and UAA , and being chosen either in the same reading frame as the open reading frame coding said protein, or in a reading frame shifted by one or two nucleotides relative to the open reading frame coding said protein, and said cPEP being capable to modulate the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein, said protein being encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1 , Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arrô, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3 , Del 1, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gape, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1 , Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1 , Spt, Stn8, Tap46, Topp6, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 and Wus.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. In one embodiment, the invention relates to the method as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the method as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. Dans un mode de réalisation, l’invention concerne également le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385. In one embodiment, the invention also relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un autre mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est choisi parmi les séquences : SEQ ID NOs : 162 à 206, 404 et 406 à 417. In another embodiment, the invention relates to the method as described above, in which said cPEP is chosen from the sequences: SEQ ID NOs: 162 to 206, 404 and 406 to 417.
En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est choisi parmi les séquences : SEQ ID NO : 162, SEQ ID NO : 163 et SEQ ID NOs : 164 à 174. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est de séquence SEQ ID NO : 162. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est de séquence : SEQ ID NO : 163. En particulier, l’invention concerne l’utilisation d’un cPEP telle que décrite précédemment, dans laquelle ledit cPEP est choisi parmi les séquences : SEQ ID NOs : 164 à 174. In particular, the invention relates to the method as described above, in which said cPEP is chosen from the sequences: SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NOs: 164 to 174. In particular, the invention concerns relates to the method as described above, in which said cPEP is of sequence SEQ ID NO: 162. In particular, the invention relates to the method as described previously, in which said cPEP is of sequence: SEQ ID NO: 163. in particular, the invention relates to the use of a cPEP as described above, in which said cPEP is chosen from the sequences: SEQ ID NOs: 164 to 174.
Dans un autre mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est impliquée dans au moins un phénotype de plante choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’introduction dudit cPEP entraîne une précocité de la montaison chez ladite plante. In another embodiment, the invention relates to the method as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells. In one embodiment, the invention relates to the method as described above, in which the introduction of said cPEP results in early bolting in said plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’introduction dudit cPEP entraîne une précocité de la floraison chez ladite plante. In one embodiment, the invention relates to the method as described above, in which the introduction of said cPEP results in earlier flowering in said plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’introduction dudit cPEP entraîne une augmentation de la taille de la tige chez ladite plante. In one embodiment, the invention relates to the method as described above, in which the introduction of said cPEP causes an increase in the size of the stem in said plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’introduction dudit cPEP entraîne une précocité de croissance de la tige chez ladite plante. In one embodiment, the invention relates to the method as described above, in which the introduction of said cPEP results in earlier growth of the stem in said plant.
Les Inventeurs ont en effet constaté de manière inattendue qu’il est possible d’appliquer directement un cPEP sur la plante, e.g. via l’emploi de la composition de l’invention (cf. supra) comprenant un cPEP, pour moduler l’accumulation d’une protéine cible dans la plante, ce qui indique que le cPEP est capté par la plante. The inventors have in fact unexpectedly observed that it is possible to directly apply a cPEP to the plant, e.g. via the use of the composition of the invention (see above) comprising a cPEP, to modulate the accumulation of a target protein in the plant, indicating that cPEP is taken up by the plant.
Par conséquent, dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit cPEP est introduit dans ladite plante : par arrosage, par pulvérisation ou par l’ajout d’un engrais, d’un terreau, d’un substrat de culture ou d’un support en contact avec la plante, ledit cPEP étant notamment administré à la plante sous la forme d’une composition comprenant de IO’9 M à IO’4 M dudit cPEP ; par arrosage, par trempage, par pulvérisation ou par l’ajout d’un engrais, d’un terreau, d’un substrat de culture ou d’un support en contact avec la plante, ledit cPEP étant notamment administré à une graine ou une semence sous la forme d’une composition comprenant de 10'9 M à 10'4 M dudit cPEP ; ou par le biais d’un acide nucléique codant ledit cPEP et comprenant les moyen d’exprimer ledit cPEP, ledit acide nucléique étant introduit artificiellement dans la plante. Consequently, in one embodiment, the invention relates to the method as described above, in which said cPEP is introduced into said plant: by watering, by spraying or by adding a fertilizer, a potting soil, a culture substrate or a support in contact with the plant, said cPEP being in particular administered to the plant in the form of a composition comprising from IO' 9 M to IO' 4 M of said cPEP; by watering, by soaking, by spraying or by adding a fertilizer, a potting soil, a growing substrate or a support in contact with the plant, said cPEP being in particular administered to a seed or a seed in the form of a composition comprising from 10'9 M to 10'4 M of said cPEP; or by means of a nucleic acid encoding said cPEP and comprising the means of expressing said cPEP, said nucleic acid being artificially introduced into the plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel ledit cPEP est introduit artificiellement par voie externe dans la plante, de préférence par arrosage, par pulvérisation ou par l’ajout d’un engrais, d’un terreau, d’un substrat de culture ou d’un support inerte. Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel ledit cPEP est introduit par arrosage. In one embodiment, the invention relates to the method as defined above, in which said cPEP is artificially introduced externally into the plant, preferably by watering, by spraying or by the addition of a fertilizer, potting soil, a growing medium or an inert support. In one embodiment, the invention relates to the method as defined above, in which said cPEP is introduced by watering.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel ledit cPEP est introduit par pulvérisation. In one embodiment, the invention relates to the method as defined above, in which said cPEP is introduced by spraying.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel ledit cPEP est introduit par l’ajout d’un engrais. In one embodiment, the invention relates to the process as defined above, in which said cPEP is introduced by the addition of a fertilizer.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel la plante est traitée avec une composition comprenant de 10'9 M à 10'4 M dudit cPEP, ou comprenant notamment 10'9 M, 10'8 M, 10'7 M, 10'6 M, 10'5 M ou 10'4 M dudit cPEP. De préférence, les compositions ont une concentration de 10'8 M à 10'5 M pour une application par arrosage ou par pulvérisation sur la plante. In one embodiment, the invention relates to the process as defined above, in which the plant is treated with a composition comprising from 10' 9 M to 10' 4 M of said cPEP, or comprising in particular 10' 9 M, 10' 8 M, 10' 7 M, 10' 6 M, 10' 5 M or 10' 4 M of said cPEP. Preferably, the compositions have a concentration of 10'8 M to 10'5 M for application by watering or spraying on the plant.
De manière complémentaire, des compositions plus ou moins concentrées peuvent être envisagées pour traiter la plante avec le cPEP. Par exemple, et de manière non limitative, des compositions plus concentrées comprenant de 10'1 M à 10'3 M, ou comprenant notamment 10' 2 M de cPEP, peuvent être utilisées dans le cas où le cPEP introduit artificiellement par voie externe est administré à la plante par épandage. In addition, more or less concentrated compositions can be considered to treat the plant with cPEP. For example, and in a non-limiting manner, more concentrated compositions comprising from 10' 1 M to 10' 3 M, or comprising in particular 10' 2 M of cPEP, can be used in the case where the cPEP artificially introduced externally is administered to the plant by spreading.
Dans un autre aspect, l’invention ci-dessus concerne une plante modifiée contenant un cPEP, laquelle « plante modifiée » correspond à une plante dans laquelle a été introduit artificiellement un cPEP, notamment par arrosage, par pulvérisation ou via un engrais. In another aspect, the above invention relates to a modified plant containing a cPEP, which “modified plant” corresponds to a plant into which a cPEP has been artificially introduced, in particular by watering, by spraying or via fertilizer.
Dans un mode de réalisation, l’invention concerne la plante modifiée comprenant un cPEP introduit par voie exogène, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In one embodiment, the invention relates to the modified plant comprising a cPEP introduced exogenously, said cPEP having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said cPEP being capable of modulate the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the modified plant as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; Or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne donc la plante modifiée telle que décrite précédemment, dans laquelle ledit fragment comprend un codon initiateur AUG codant une méthionine initiatrice et est dépourvu d’un codon STOP choisi parmi les codons : UAG, UGA et UAA. L’invention concerne également la plante modifiée telle que décrite précédemment, dans laquelle ledit fragment est dépourvu d’un codon initiateur AUG codant une méthionine initiatrice et comprend un codon STOP choisi parmi les codons : UAG, UGA et UAA. In one embodiment, the invention therefore relates to the modified plant as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA . The invention also relates to the modified plant as described above, in which said fragment is devoid of an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
Dans un mode de réalisation, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; et d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the modified plant as described above, in which said fragment is devoid of: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans le même cadre de lecture que le cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans le cadre de lecture déterminé par le codon d’initiation du cadre ouvert de lecture de ladite protéine. In one embodiment, the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the naturally occurring nucleic acid sequence. translated into said plant cell. In other words, the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
Dans un mode de réalisation, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally translated nucleic acid sequence. in said plant cell. In particular, the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the plant modified as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides at 3' (or by one nucleotide at 5') relative to the open reading frame of the sequence of nucleic acids naturally translated in said plant cell. In other words, the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in the 5') or two nucleotides in the 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne une plante modifiée comprenant un cPEP introduit par voie exogène, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit fragment ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ladite séquence d’acides nucléiques comprenant deux parties contiguës : une partie située au sein d’une séquence d’acides nucléiques réputée non codante (/.e. non traduite naturellement, e.g. 3’IITR ou 5’IITR) ; et une partie située au sein d’une séquence d’acides nucléiques réputée codante (/.e. traduite naturellement, e.g. exon) ; ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In one embodiment, the invention relates to a modified plant comprising an exogenously introduced cPEP, said cPEP having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, and said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/.e. not naturally translated, e.g. 3'IITR or 5'IITR); and a part located within a nucleic acid sequence known to be coding (/e. naturally translated, e.g. exon); said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante modifiée telle que décrite précédemment, ladite plante appartenant à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacusAmaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). Dans un autre aspect, l’invention ci-dessus concerne une plante transgénique comprenant un acide nucléique codant un cPEP et les moyens de l’exprimer, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In one embodiment, the invention relates to the modified plant as described above, said plant belonging to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacusAmaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp . (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn). In another aspect, the above invention relates to a transgenic plant comprising a nucleic acid encoding a cPEP and the means for expressing it, said cPEP having a size of from 4 to 70 amino acids, in particular from 4 to 41 acids. amino, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of an encoded protein by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; ou d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the transgenic plant as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; or a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne donc la plante transgénique telle que décrite précédemment, dans laquelle ledit fragment comprend un codon initiateur AUG codant une méthionine initiatrice et est dépourvu d’un codon STOP choisi parmi les codons : UAG, UGA et UAA. L’invention concerne également la plante transgénique telle que décrite précédemment, dans laquelle ledit fragment est dépourvu d’un codon initiateur AUG codant une méthionine initiatrice et comprend un codon STOP choisi parmi les codons : UAG, UGA et UAA. In one embodiment, the invention therefore relates to the transgenic plant as described above, in which said fragment comprises an AUG initiator codon encoding an initiator methionine and is devoid of a STOP codon chosen from the codons: UAG, UGA and UAA . The invention also relates to the transgenic plant as described above, in which said fragment lacks an AUG initiator codon encoding an initiator methionine and comprises a STOP codon chosen from the codons: UAG, UGA and UAA.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle ledit fragment est dépourvu : du codon initiateur AUG codant une méthionine initiatrice ; et d’un codon STOP choisi parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi : soit dans le même cadre ouvert de lecture que celui codant ladite protéine ; soit dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the transgenic plant as described above, in which said fragment lacks: the AUG initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen: either in the same open reading frame as that encoding said protein; either in an open reading frame shifted by one or two nucleotides compared to that encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans le même cadre de lecture que le cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans le cadre de lecture déterminé par le codon d’initiation du cadre ouvert de lecture de ladite protéine. In one embodiment, the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in the same reading frame as the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in the reading frame determined by the initiation codon of the open reading frame of said protein.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally translated nucleic acid sequence. in said plant cell. In particular, the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in the 5') or two nucleotides in the 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne une plante transgénique comprenant un cPEP introduit par voie exogène, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 70 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit fragment ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ladite séquence d’acides nucléiques comprenant deux parties contiguës : une partie située au sein d’une séquence d’acides nucléiques réputée non codante (/.e. non traduite naturellement, e.g. 3’IITR ou 5’IITR) ; et une partie située au sein d’une séquence d’acides nucléiques réputée codante (/.e. traduite naturellement, e.g. exon) ; ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In one embodiment, the invention relates to a transgenic plant comprising a cPEP introduced exogenously, said cPEP having a size of from 4 to 70 amino acids, in particular from 4 to 70 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said fragment having a size of 3n nucleotides, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, and said nucleic acid sequence comprising two contiguous parts: a part located within a nucleic acid sequence deemed non-coding (/.e. not naturally translated, eg 3'IITR or 5'IITR); and a part located within a nucleic acid sequence known to be coding (/e. naturally translated, eg exon); said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que définie précédemment, dans laquelle la séquence codant ledit cPEP est plus courte que la séquence de l’ARNm codant ladite protéine. In one embodiment, the invention relates to the transgenic plant as defined above, in which the sequence encoding said cPEP is shorter than the sequence of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, ladite plante appartenant à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus , Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the transgenic plant as described above, said plant belonging to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle l’expression dudit cPEP est placée sous le contrôle d’un promoteur fort, de préférence un promoteur fort constitutif tel que le promoteur 35S. In one embodiment, the invention relates to the transgenic plant as described above, in which the expression of said cPEP is placed under the control of a strong promoter, preferably a strong constitutive promoter such as the 35S promoter.
A tout égard, il convient de noter que les différents aspects de l’invention No. 1 , tout comme les différents modes de réalisation de celle-ci sont interdépendants. Ces derniers peuvent donc être combinés entre eux pour obtenir des aspects et/ou des modes de réalisation préférés de l’invention No. 1 non explicitement décrits. Ceci est également valable pour l’ensemble des définitions fourni dans la présente description, lequel s’applique à tous les aspects de l’invention No. 1 et ses modes de réalisation. Considérant la seconde invention (altPEP), un premier aspect de celle-ci concerne un procédé de préparation et de détermination d’un altPEP, ledit altPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés ; étant capable de moduler l’accumulation d’une protéine dans une cellule végétale ; et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, ledit procédé comprenant : a. une étape de détermination de la séquence d’acides nucléiques de l’ARN messager (ARNm) codant ladite protéine ; b. une étape de détermination au sein de cet ARNm de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine ; c. une étape de détermination au sein de cette séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale d’un fragment naturellement traduit de celle-ci, ledit fragment ayant une taille de 3n nucléotides susceptible d’être traduite via le code génétique en un peptide, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ledit fragment ayant une taille inférieure à celle de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale ; d. une étape de production dudit peptide ; et e. une étape de comparaison : In all respects, it should be noted that the different aspects of invention No. 1, just like the different embodiments thereof, are interdependent. The latter can therefore be combined with each other to obtain preferred aspects and/or embodiments of invention No. 1 not explicitly described. This is also valid for all definitions provided in this description, which applies to all aspects of Invention No. 1 and its embodiments. Considering the second invention (altPEP), a first aspect thereof relates to a method for preparing and determining an altPEP, said altPEP: having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids ; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a. a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein; b. a step of determining within this mRNA the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein; vs. a step of determining within this nucleic acid sequence naturally translated in said plant cell a naturally translated fragment thereof, said fragment having a size of 3n nucleotides capable of being translated via the genetic code into a peptide , n being between 4 and 70, in particular n being between 4 and 41, and said fragment having a size smaller than that of the nucleic acid sequence naturally translated in said plant cell; d. a step of producing said peptide; summer. a comparison step:
- entre l’accumulation de ladite protéine dans une cellule végétale en présence dudit peptide et l’accumulation de ladite protéine dans une cellule végétale de même type en absence dudit peptide ; et/ou - between the accumulation of said protein in a plant cell in the presence of said peptide and the accumulation of said protein in a plant cell of the same type in the absence of said peptide; and or
- entre le phénotype d’une plante en présence dudit peptide et le phénotype d’une plante de même type en absence dudit peptide, dans laquelle : - between the phenotype of a plant in the presence of said peptide and the phenotype of a plant of the same type in the absence of said peptide, in which:
- une différence de la quantité de ladite protéine en présence dudit peptide par rapport à la quantité de ladite protéine en absence dudit peptide ; et/ou - a difference in the quantity of said protein in the presence of said peptide compared to the quantity of said protein in the absence of said peptide; and or
- une différence du phénotype en présence dudit peptide par rapport au phénotype en absence dudit peptide, indique que ledit peptide est un altPEP capable de moduler l’accumulation de ladite protéine dans une cellule végétale. La présente invention repose sur la constatation inattendue faite par les Inventeurs qu’il est possible de moduler spécifiquement l’accumulation d’une protéine à l’aide d’un peptide particulier produit naturellement, dont la séquence correspond à la traduction d’un fragment de l’ARN messager (ARNm) codant ladite protéine, ledit fragment étant choisi sur la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine. - a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is an altPEP capable of modulating the accumulation of said protein in a plant cell. The present invention is based on the unexpected observation made by the inventors that it is possible to specifically modulate the accumulation of a protein using a particular peptide produced naturally, the sequence of which corresponds to the translation of a fragment messenger RNA (mRNA) encoding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
Dans l’invention, le terme « altPEP » (alternative peptide) désigne un peptide capable de moduler spécifiquement l’accumulation d’une protéine une fois introduit dans une cellule végétale. In the invention, the term “altPEP” (alternative peptide) designates a peptide capable of specifically modulating the accumulation of a protein once introduced into a plant cell.
Selon l’invention, un altPEP est présent naturellement dans une cellule végétale. Cela signifie que la cellule végétale contient l’information de l’altPEP et les moyens de permettre son expression (/.e. codon START et codon STOP). According to the invention, an altPEP is naturally present in a plant cell. This means that the plant cell contains the altPEP information and the means to allow its expression (/.e. START codon and STOP codon).
Un altPEP est peut donc être présent dans une cellule végétale et la quantité de celui-ci peut être modifiée par l’ajout artificiel de celui-ci, sous la forme d’un peptide ou sous la forme d’un acide nucléique codant ledit peptide, dans la cellule végétale. An altPEP may therefore be present in a plant cell and the quantity thereof may be modified by the artificial addition of it, in the form of a peptide or in the form of a nucleic acid encoding said peptide. , in the plant cell.
La spécificité de l’altPEP vis-à-vis d’une protéine cible (d’un gène cible) est déterminée par sa séquence d’acides aminés. En effet, la séquence d’un altPEP correspond à la traduction d’un fragment de l’ARNm codant ladite protéine, ledit fragment étant choisi sur la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine. The specificity of altPEP towards a target protein (target gene) is determined by its amino acid sequence. Indeed, the sequence of an altPEP corresponds to the translation of a fragment of the mRNA encoding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein.
La séquence peptidique d’un altPEP peut donc être déterminée à partir d’un fragment de l’ARNm codant ladite protéine. The peptide sequence of an altPEP can therefore be determined from a fragment of the mRNA encoding said protein.
Dans l’invention, le fragment de l’ARNm utilisé pour déterminer la séquence de l’altPEP peut être choisi dans les deux autres cadres de lecture que celui codant la protéine dont on souhaite moduler l’accumulation existants sur la séquence de l’ARNm. En d’autres termes, un fragment peut être sélectionné dans les cadres de lecture +2 ou +3. Sur ce point, il est possible que les deux autres des cadres de lecture (le +2 et le +3) contiennent l’information d’un altPEP comme il est possible que seulement un des deux des cadres de lecture (le +2 ou le +3) contiennent l’information d’un altPEP. In the invention, the fragment of the mRNA used to determine the sequence of altPEP can be chosen in the two other reading frames than that coding the protein whose accumulation it is desired to modulate existing on the sequence of the mRNA . In other words, a fragment can be selected in reading frames +2 or +3. On this point, it is possible that the other two reading frames (+2 and +3) contain the information of an altPEP as it is possible that only one of the two reading frames (+2 or +3) contain the information of an altPEP.
Dans l’invention, le terme « cadre de lecture » désigne le regroupement des nucléotides constituant une séquence d’acides nucléiques en triplets (ou codons) consécutifs, qui se succèdent sans interruption ni recouvrement. D’une manière générale, les altPEPs ont une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, notamment une taille comprise de 5 à 40 acides aminés, de 7 à 20 acides aminés ou plus particulièrement une taille comprise de 8 à 15 acides aminés. Par conséquent, la séquence d’un altPEP correspond à la traduction d’un fragment compris de 4 à 41 triplets de nucléotides, notamment d’un fragment compris de 5 à 40 triplets de nucléotides, de 7 à 20 triplets de nucléotides ou plus particulièrement d’un fragment compris de 8 à 15 triplets de nucléotides. In the invention, the term “reading frame” designates the grouping of nucleotides constituting a sequence of nucleic acids into consecutive triplets (or codons), which follow one another without interruption or overlap. Generally speaking, altPEPs have a size of 4 to 70 amino acids, in particular of 4 to 41 amino acids, in particular a size of 5 to 40 amino acids, of 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids. Consequently, the sequence of an altPEP corresponds to the translation of a fragment comprising 4 to 41 nucleotide triplets, in particular a fragment comprising 5 to 40 nucleotide triplets, 7 to 20 nucleotide triplets or more particularly of a fragment comprising 8 to 15 nucleotide triplets.
En d’autres termes, la séquence d’un altPEP correspond à la traduction en acides aminés d’un fragment de « 3n » nucléotides de l’ARNm de la protéine cible, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , notamment compris de 5 à 40, de 7 à 20 ou plus particulièrement compris de 8 à 15. In other words, the sequence of an altPEP corresponds to the translation into amino acids of a fragment of “3n” nucleotides of the mRNA of the target protein, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, in particular from 5 to 40, from 7 to 20 or more particularly from 8 to 15.
Par exemple, si n est égal à 5, l’altPEP a une taille de 5 acides aminés et correspond à la traduction d’un fragment de 15 (= 3 x 5) nucléotides. Par exemple, si n est égal à 40, l’altPEP a une taille de 40 acides aminés et correspond à la traduction d’un fragment de 120 (= 3 x 40) nucléotides. Par exemple, si n est égal à 70, l’altPEP a une taille de 70 acides aminés et correspond à la traduction d’un fragment de 210 (= 3 x 70) nucléotides. Etc. For example, if n is equal to 5, altPEP has a size of 5 amino acids and corresponds to the translation of a fragment of 15 (= 3 x 5) nucleotides. For example, if n is equal to 40, altPEP has a size of 40 amino acids and corresponds to the translation of a fragment of 120 (= 3 x 40) nucleotides. For example, if n is equal to 70, altPEP has a size of 70 amino acids and corresponds to the translation of a fragment of 210 (= 3 x 70) nucleotides. Etc.
Les altPEPs ont une taille de 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22,The altPEPs have a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 ou 70 acides aminés, et correspondent respectivement à la traduction de fragments de 12, 15, 18, 21 , 24,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids, and correspond respectively to the translation of fragments of 12, 15, 18, 21, 24,
27, 30, 33, 36, 39, 42, 45, 48, 51 , 54, 57, 60, 63, 66, 69, 72, 75, 78, 81 , 84, 87, 90, 93, 96, 99,27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99,
102, 105, 108, 111 , 114, 117, 123, 126, 129, 132, 135, 138, 141 , 144, 147, 150, 153, 156, 159, 162, 165, 168, 171 , 174, 177, 180, 183, 186, 189, 192, 195, 198, 201 , 204, 207 ou 210 nucléotides. 102, 105, 108, 111, 114, 117, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207 or 210 nucleotides.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ledit fragment a une taille de 3n nucléotides, n étant compris : de 4 à 41 ; de 5 à 40 ; de 7 à 20 ; ou de 8 à 15. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said fragment has a size of 3n nucleotides, n being included: from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ledit peptide a une taille choisie parmi : 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 et 70 acides aminés. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said peptide has a size chosen from: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 and 70 amino acids.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ledit altPEP a une taille inférieure à celle de ladite protéine (/.e. celle dont l’altPEP module l’accumulation). In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said altPEP has a size smaller than that of said protein (/e. that of which altPEP modulates accumulation).
Comme indiqué plus haut, les altPEPs ont la capacité de moduler spécifiquement l’accumulation d’une protéine sans que l’accumulation de l’ARNm lui correspondant soit impacté. Autrement dit, l’ajout d’un altPEP dans une cellule végétale ne modifie pas la quantité d’ARNm permettant d’exprimer la protéine qu’il a la capacité de réguler, mais seulement la quantité de ladite protéine. As indicated above, altPEPs have the capacity to specifically modulate the accumulation of a protein without the accumulation of the corresponding mRNA being impacted. In other words, the addition of an altPEP in a plant cell does not modify the quantity of mRNA allowing the expression of the protein that it has the capacity to regulate, but only the quantity of said protein.
Selon l’invention, le terme « protéine » désigne une séquence d’acides aminés dont l’information est codée par un gène présent sur le génome d’une cellule végétale. Par « gène », on désigne donc, notamment, la séquence d’acides nucléiques nécessaire à la synthèse de ladite protéine. Aussi, un gène comprend davantage que les nucléotides codant la séquence d’acides aminés de la protéine. Par exemple, un gène inclut les séquences d’ADN nécessaires à la synthèse d’un pré-messager (pré-ARNm), lequel est ensuite maturé par la machinerie cellulaire en un ARN messager (ARNm). Ce dernier peut alors être traduit, via les ribosomes, en une protéine. According to the invention, the term “protein” designates a sequence of amino acids whose information is encoded by a gene present on the genome of a plant cell. By “gene”, we therefore designate, in particular, the nucleic acid sequence necessary for the synthesis of said protein. Also, a gene includes more than the nucleotides encoding the amino acid sequence of the protein. For example, a gene includes the DNA sequences necessary for the synthesis of a pre-messenger (pre-mRNA), which is then processed by the cellular machinery into messenger RNA (mRNA). The latter can then be translated, via ribosomes, into a protein.
Au vu de ce qui précède, on comprend que l’ARN pré-messager (pré-ARNm) n’a pas subi d’épissage et est susceptible de contenir des introns, tandis que l’ARN messager (ARNm) mature peut avoir subi un épissage et ne contient que des exons. In view of the above, it is understood that pre-messenger RNA (pre-mRNA) has not undergone splicing and is likely to contain introns, while mature messenger RNA (mRNA) may have undergone a splice and contains only exons.
Pour préparer un altPEP capable de moduler l’accumulation d’une protéine, il convient de traduire un fragment de l’ARNm codant ladite protéine, ledit fragment étant choisi sur la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine, lequel ne comprend ni la région 5’-UTR, ni la région 3’-UTR de l’ARNm. To prepare an altPEP capable of modulating the accumulation of a protein, it is appropriate to translate a fragment of the mRNA encoding said protein, said fragment being chosen from the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein, which includes neither the 5'-UTR region nor the 3'-UTR region of the mRNA.
Dans l’invention, la « modulation » de l’accumulation d’une protéine désigne soit une augmentation de l’accumulation de ladite protéine (/.e. une augmentation de la quantité de protéine dans la cellule végétale), soit une diminution de l’accumulation de ladite protéine (/.e. une diminution de la quantité de protéine dans la cellule végétale). Autrement dit, un mode de réalisation de l’invention a pour objet le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite modulation de l’accumulation de ladite protéine induite par ledit altPEP est : une diminution de l’accumulation de ladite protéine ; ou une augmentation de l’accumulation de ladite protéine. In the invention, the “modulation” of the accumulation of a protein designates either an increase in the accumulation of said protein (/e. an increase in the quantity of protein in the plant cell), or a decrease in the accumulation of said protein (/e. a decrease in the quantity of protein in the plant cell). In other words, one embodiment of the invention relates to the method of preparing and determining a altPEP as described above, wherein said modulation of the accumulation of said protein induced by said altPEP is: a decrease in the accumulation of said protein; or an increase in the accumulation of said protein.
L’augmentation et la diminution de l’accumulation de ladite protéine peuvent être mesurées et suivies à l’aide de méthodes bien connues de l’homme du métier, telles que le couplage de la protéine à un marqueur via l’utilisation de cassettes d’expression particulières, ou un Western blot. The increase and decrease in the accumulation of said protein can be measured and monitored using methods well known to those skilled in the art, such as the coupling of the protein to a marker via the use of cassettes. particular expression, or a Western blot.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel à l’étape e., la quantité de protéine en présence dudit peptide est supérieure à la quantité de protéine en absence dudit peptide. En d’autres termes, en présence d’un altPEP favorisant l’augmentation de l’accumulation de la protéine, la traduction de l’ARNm correspondant est augmentée, ce qui conduit à une production plus importante de la protéine sans que soit modifiée la quantité dudit ARNm. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which in step e., the quantity of protein in the presence of said peptide is greater than the quantity of protein in the absence of said peptide. In other words, in the presence of an altPEP promoting increased accumulation of the protein, the translation of the corresponding mRNA is increased, which leads to greater production of the protein without modifying the quantity of said mRNA.
Dans un autre mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel à l’étape e., la quantité de protéine en présence dudit peptide est inférieure à la quantité de protéine en absence dudit peptide. En d’autres termes, en présence d’un altPEP favorisant la diminution de l’accumulation de la protéine, la traduction de l’ARNm correspondant est diminuée (inhibée), ce qui conduit à une production moins importante de la protéine sans que soit modifiée la quantité dudit ARNm. In another embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which in step e., the quantity of protein in the presence of said peptide is less than the quantity of protein in the absence of said peptide. In other words, in the presence of an altPEP promoting the reduction of the accumulation of the protein, the translation of the corresponding mRNA is reduced (inhibited), which leads to less production of the protein without either modified the quantity of said mRNA.
Dans l’invention, le fragment de l’ARNm codant ledit altPEP est situé au sein la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine, et ledit fragment en tant que tel peut également être traduit naturellement dans ladite cellule végétale. Ceci, quel que soit le cadre de lecture utilisé (/.e. le +2 et/ou le +3). L’existence d’un altPEP au sein de ladite cellule végétale est donc naturelle et a pour origine soit la machinerie cellulaire, soit une action de l’Homme. Pour cela, il est possible soit d’introduire artificiellement ledit altPEP en tant que tel, soit d’introduire une cassette d’expression comprenant la séquence d’acides nucléiques codant ledit altPEP et les moyens de l’exprimer dans ladite cellule végétale. In the invention, the fragment of the mRNA encoding said altPEP is located within the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein, and said fragment as such can also be translated naturally in said plant cell. This, regardless of the reading frame used (e.g. +2 and/or +3). The existence of an altPEP within said plant cell is therefore natural and originates either from cellular machinery or from human action. For this, it is possible either to artificially introduce said altPEP as such, or to introduce an expression cassette comprising the nucleic acid sequence encoding said altPEP and the means of expressing it in said plant cell.
Dans l’invention, la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale, laquelle comprend un fragment portant l’information d’un altPEP, est une région de l’ARNm qualifiée de « codante », c’est-à-dire qu’elle correspond à une région de l’ARNm qui code tout ou partie de la protéine fonctionnelle. Cette séquence correspond donc au cadre ouvert de lecture principal (/.e. le +1), lequel code la protéine dont on veut moduler l’accumulation. In the invention, the nucleic acid sequence naturally translated in said plant cell, which comprises a fragment carrying the information of an altPEP, is a region of the mRNA described as “coding”, that is to say that it corresponds to a region of the mRNA which codes all or part of the functional protein. This sequence therefore corresponds to the main open reading frame (/.e. the +1), which codes the protein whose accumulation we want to modulate.
Dans l’invention, les termes « cadre ouvert de lecture » et « ORF » (open reading frame) sont équivalents, et peuvent être utilisés l’un pour l’autre. Ils correspondent à une séquence de nucléotides (acides nucléiques) dans une molécule d’ADN ou d’ARN pouvant potentiellement coder un peptide ou une protéine : ledit cadre ouvert de lecture débute par un codon START (le codon START codant généralement une méthionine), suivi d’une série de codons (chaque codon codant un acide aminé), et se termine par un codon STOP (le codon STOP n’étant pas traduit). In the invention, the terms “open reading frame” and “ORF” (open reading frame) are equivalent, and can be used interchangeably. They correspond to a sequence of nucleotides (nucleic acids) in a DNA or RNA molecule that can potentially encode a peptide or a protein: said open reading frame begins with a START codon (the START codon generally encoding a methionine), followed by a series of codons (each codon encoding an amino acid), and ends with a STOP codon (the STOP codon not being translated).
La région codante de l’ARNm correspond donc à la séquence génétique délimitée par le codon START ou codon d’initiation (codant le plus souvent une méthionine) à l’extrémité 5’ et par le codon STOP à l’extrémité 3’. La région codante de l’ARNm ne comprend pas donc les séquences introniques éventuellement présentes dans la séquence d’un gène ou de l’ARN pré-messager (pré-ARNm), ni les régions 5’UTR et 3’UTR, car celles-ci ne sont pas traduites et ne codent donc pas une partie de la protéine fonctionnelle du gène. The coding region of the mRNA therefore corresponds to the genetic sequence delimited by the START codon or initiation codon (most often encoding a methionine) at the 5' end and by the STOP codon at the 3' end. The coding region of the mRNA therefore does not include the intronic sequences possibly present in the sequence of a gene or pre-messenger RNA (pre-mRNA), nor the 5'UTR and 3'UTR regions, because those -these are not translated and therefore do not encode part of the functional protein of the gene.
Dans l’invention, la séquence d’un altPEP est déterminée en effectuant une traduction d’un fragment de l’ARNm de la protéine dont on veut moduler l’accumulation, ledit fragment étant choisi sur la séquence (codante) d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine. Aussi, une même séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale peut donner des altPEPs différents selon le fragment de l’ARNm choisi. Par ailleurs, ce même fragment d’ARNm peut lui aussi donner des altPEPs différents selon le cadre de lecture utilisé pour le traduire, /.e. selon le regroupement des nucléotides de la séquence en triplets consécutifs. En effet et comme précédemment mentionné, une traduction peut être réalisée dans deux cadres de lecture différents (le +2 et/ou le +3) conduisant ainsi à potentiellement deux altPEPs différents. In the invention, the sequence of an altPEP is determined by carrying out a translation of a fragment of the mRNA of the protein whose accumulation we want to modulate, said fragment being chosen from the (coding) sequence of nucleic acids naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein. Also, the same nucleic acid sequence naturally translated in said plant cell can give different altPEPs depending on the mRNA fragment chosen. Furthermore, this same mRNA fragment can also give different altPEPs depending on the reading frame used to translate it, /.e. according to the grouping of the nucleotides of the sequence into consecutive triplets. Indeed and as previously mentioned, a translation can be carried out in two different reading frames (+2 and/or +3) thus potentially leading to two different altPEPs.
Selon l’invention, le cadre de lecture “+1” correspond au cadre de lecture déterminé par le codon d’initiation de la protéine, /.e. le codon START du cadre ouvert de lecture utilisé naturellement pour la traduction de l’ARNm. According to the invention, the “+1” reading frame corresponds to the reading frame determined by the initiation codon of the protein, /.e. the START codon of the open reading frame used naturally for mRNA translation.
Les cadres de lecture “+2” et “+3” correspondent à des cadres de lecture qui ne sont pas ou peu utilisés naturellement pour la traduction de l’ARNm. Selon l’invention, le cadre de lecture “+2” correspond au cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture utilisé naturellement pour la traduction de l’ARNm et de la protéine qu’il code. Selon l’invention, le cadre de lecture “+3” correspond au cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture utilisé naturellement pour la traduction de l’ARNm et de la protéine qu’il code. The “+2” and “+3” reading frames correspond to reading frames which are not or only rarely used naturally for mRNA translation. According to the invention, the reading frame “+2” corresponds to the reading frame shifted by one nucleotide in 3' (or two nucleotides in 5') compared to the open reading frame used naturally for the translation of the mRNA and the protein it codes . According to the invention, the “+3” reading frame corresponds to the reading frame shifted by two nucleotides at 3' (or one nucleotide at 5') relative to the open reading frame used naturally for the translation of the mRNA and the protein it encodes.
Généralement, dans le cas d’un fragment d’ARNm correspondant à une région codante et traduit selon le cadre de lecture +2 ou +3, les altPEPs obtenus possèdent une séquence différente de celle d’un fragment de la séquence d’acides aminés de la protéine naturellement codée par ledit ARNm. Generally, in the case of an mRNA fragment corresponding to a coding region and translated according to the +2 or +3 reading frame, the altPEPs obtained have a sequence different from that of a fragment of the amino acid sequence of the protein naturally encoded by said mRNA.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ledit fragment comprend : un codon initiateur codant une méthionine initiatrice ; et un codon STOP choisie parmi les codons : UAG, UGA et UAA, et dans lequel ledit fragment est choisi dans un cadre de lecture décalé d’un ou deux nucléotides par rapport au cadre ouvert de lecture codant ladite protéine. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and in which said fragment is chosen in a reading frame shifted by one or two nucleotides relative to the open reading frame encoding said protein.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ledit altPEP est un peptide hydrophobe ou un peptide hydrophile. En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ledit altPEP est un peptide hydrophobe. Par « peptide hydrophobe », on entend un peptide dont la séquence en acides aminés comprend plus de 50 % d’acides aminés hydrophobes. Par « plus de 50 % », on entend que la séquence en acides aminés comprend plus de 55 %, plus de 60 %, plus de 65 %, plus de 70 %, plus de 75 % ou plus de 80 % d’acides aminés hydrophobes. Par « plus de 50 % », on entend également que la séquence en acides aminés comprend au moins 51 %, au moins 56 %, au moins 61 %, au moins 66 %, au moins 71 %, au moins 76 % ou au moins 81 % d’acides aminés hydrophobes. Par « acides aminés hydrophobes », on entend les acides aminés choisis parmi : alanine (Ala / A), isoleucine (Ile / 1), leucine (Leu / L), méthionine (Met/ M), phénylalanine (Phe / F), tryptophane (Trp / W), tyrosine (Tyr/ Y) et valine (Val / V). In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or of two nucleotides 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the method for preparing and determining an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in 3' (or of a nucleotide at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the method for preparing and determining an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5 ') or two nucleotides 3' (or one nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said altPEP is a hydrophobic peptide or a hydrophilic peptide. In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said altPEP is a hydrophobic peptide. By “hydrophobic peptide” is meant a peptide whose amino acid sequence comprises more than 50% hydrophobic amino acids. By “more than 50%”, we mean that the amino acid sequence comprises more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80% of amino acids. hydrophobic. By “more than 50%”, we also mean that the amino acid sequence comprises at least 51%, at least 56%, at least 61%, at least 66%, at least 71%, at least 76% or at least 81% hydrophobic amino acids. By “hydrophobic amino acids” is meant the amino acids chosen from: alanine (Ala / A), isoleucine (Ile / 1), leucine (Leu / L), methionine (Met / M), phenylalanine (Phe / F), tryptophan (Trp/W), tyrosine (Tyr/Y) and valine (Val/V).
En particulier, l’invention concerne également le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ledit altPEP est un peptide hydrophile. Par « peptide hydrophile », on entend un peptide dont la séquence en acides aminés comprend plus de 50 % d’acides aminés hydrophiles. Par « plus de 50 % », on entend que la séquence en acides aminés comprend plus de 55 %, plus de 60 %, plus de 65 %, plus de 70 %, plus de 75 % ou plus de 80 % d’acides aminés hydrophiles. Par « plus de 50 % », on entend également que la séquence en acides aminés comprend au moins 51 %, au moins 56 %, au moins 61 %, au moins 66 %, au moins 71 %, au moins 76 % ou au moins 81 % d’acides aminés hydrophiles. Par « acides aminés hydrophiles », on entend les acides aminés choisis parmi : acide aspartique (Asp / D), acide glutamique (Glu / E), arginine (Arg / R), asparagine (Asn / N), glutamine (Gin / Q), histidine (His I H), lysine (Lys / K), sérine (Ser / S) et thréonine (Thr / T). In particular, the invention also relates to the method for preparing and determining an altPEP as described above, in which said altPEP is a hydrophilic peptide. By “hydrophilic peptide” is meant a peptide whose amino acid sequence comprises more than 50% hydrophilic amino acids. By “more than 50%”, we mean that the amino acid sequence comprises more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80% of amino acids. hydrophilic. By “more than 50%”, we also mean that the amino acid sequence comprises at least 51%, at least 56%, at least 61%, at least 66%, at least 71%, at least 76% or at least 81% hydrophilic amino acids. By “hydrophilic amino acids”, we mean the amino acids chosen from: aspartic acid (Asp / D), glutamic acid (Glu / E), arginine (Arg / R), asparagine (Asn / N), glutamine (Gin / Q ), histidine (His I H), lysine (Lys/K), serine (Ser/S) and threonine (Thr/T).
Selon l’invention, un altPEP peut être produit par tout type de moyens accessibles à l’homme du métier. According to the invention, an altPEP can be produced by any type of means accessible to those skilled in the art.
De manière non limitative, un altPEP peut être produit aussi bien par synthèse, que par expression recombinante dans des systèmes homologues ou hétérologues. L’altPEP ainsi produit peut ensuite être introduit dans une cellule pour moduler l’accumulation d’une protéine cible. De manière non limitative, il est également possible de produire un altPEP directement dans la cellule végétale contenant la protéine cible, en introduisant artificiellement dans celle-ci un acide nucléique (tel qu’un vecteur d’expression) codant ledit altPEP. In a non-limiting manner, an altPEP can be produced both by synthesis and by recombinant expression in homologous or heterologous systems. The altPEP thus produced can then be introduced into a cell to modulate the accumulation of a target protein. In a non-limiting manner, it is also possible to produce an altPEP directly in the plant cell containing the target protein, by artificially introducing into it a nucleic acid (such as an expression vector) encoding said altPEP.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel, à l’étape d., la production dudit peptide est réalisée par synthèse peptidique ou par expression recombinante. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which, in step d., the production of said peptide is carried out by peptide synthesis or by recombinant expression.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel, à l’étape d., la production dudit peptide est réalisée à l’aide d’un acide nucléique codant ledit peptide introduit dans une cellule. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which, in step d., the production of said peptide is carried out using an acid nucleic acid encoding said peptide introduced into a cell.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel, à l’étape e., la production dudit peptide est réalisée à l’aide d’un acide nucléique codant ledit peptide introduit dans ladite cellule végétale ou dans ladite plante. In one embodiment, the invention relates to the process for preparing and determining an altPEP as described above, in which, in step e., the production of said peptide is carried out using an acid nucleic acid encoding said peptide introduced into said plant cell or into said plant.
Dans un mode de réalisation, l’invention concerne un procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel, à l’étape e., ledit peptide est mis en contact avec ladite cellule végétale ou dans ladite plante. In one embodiment, the invention relates to a method for preparing and determining an altPEP as described above, in which, in step e., said peptide is brought into contact with said plant cell or in said plant .
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel, à l’étape e., ledit peptide est présent dans ladite cellule végétale ou dans ladite plante suite à l’expression d’un acide nucléique codant ledit peptide dans ladite cellule végétale ou dans ladite plante. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which, in step e., said peptide is present in said plant cell or in said plant following the expression of a nucleic acid encoding said peptide in said plant cell or in said plant.
Au vu de ce qui précède, on comprend qu’un autre mode de réalisation de l’invention a pour objet le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel, à l’étape e., la présence dudit peptide dans ladite cellule végétale ou dans ladite plante résulte : In view of the above, it is understood that another embodiment of the invention relates to the method of preparing and determining an altPEP as described above, in which, in step e., the presence of said peptide in said plant cell or in said plant results:
- de l’introduction d’une séquence d’acides nucléiques codant ledit peptide et comprenant les moyens de l’exprimer ; ou - the introduction of a nucleic acid sequence encoding said peptide and comprising the means of expressing it; Or
- de l’introduction d’une séquence d’acides aminés correspondant audit peptide. - the introduction of an amino acid sequence corresponding to said peptide.
Un altPEP peut être utilisé pour moduler l’accumulation d’une protéine présente naturellement (/.e. endogène) ou non (/.e. exogène) dans ladite cellule végétale ou dans ladite plante. Une « protéine naturellement présente dans une cellule végétale ou dans une plante » correspond à une protéine endogène codée par un gène présent sur le génome de la cellule végétale ou de la plante sans qu’il y ait eu nécessité d’intervention directe ou indirecte d’un être humain. An altPEP can be used to modulate the accumulation of a protein present naturally (/e. endogenous) or not (/e. exogenous) in said plant cell or in said plant. A “protein naturally present in a plant cell or in a plant” corresponds to an endogenous protein encoded by a gene present on the genome of the plant cell or the plant without the need for direct or indirect intervention by 'a human.
Une « protéine qui n’est pas naturellement présente dans une cellule végétale ou dans une plante » correspond à une protéine exogène codée par une séquence d’acides nucléiques présente sur le génome de la cellule végétale ou de la plante qui a nécessité l’intervention d’un être humain et l’emploi de moyens connus de l’homme du métier. Une telle séquence d’acides nucléiques peut provenir de la même espèce de plante ou d’une autre espèce de plante. A “protein which is not naturally present in a plant cell or in a plant” corresponds to an exogenous protein encoded by a nucleic acid sequence present on the genome of the plant cell or plant which required the intervention of a human being and the use of means known to those skilled in the art. Such a nucleic acid sequence may come from the same plant species or from a different plant species.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est d’origine endogène dans lesdites cellules végétales ou lesdites plantes utilisées à l’étape e. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is of endogenous origin in said plant cells or said plants used in step e.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est d’origine exogène dans lesdites cellules végétales ou lesdites plantes utilisées à l’étape e., lesdites cellules végétales ou lesdites plantes utilisées à l’étape e. comprenant alors une séquence d’acides nucléiques permettant l’expression de ladite protéine. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is of exogenous origin in said plant cells or said plants used in step e., said plant cells or said plants used in step e. then comprising a nucleic acid sequence allowing the expression of said protein.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel l’accumulation de ladite protéine est déterminée via la mise en œuvre d’une technique choisie parmi : le Western blot, la mesure de l’activité enzymatique, la spectrométrie de masse et la fusion traductionnelle. En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel l’accumulation de ladite protéine est déterminée via la mise en œuvre d’un Western blot. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blot, measurement of enzyme activity, mass spectrometry and translational fusion. In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
Les Inventeurs ont constaté de manière surprenante que l’utilisation des altPEPs permet de modifier les phénotypes d’une plante visibles à l’échelle macroscopique. Il est donc tout à fait possible d’utiliser ces derniers pour affirmer (ou infirmer) que le peptide déterminé aux étapes a., b. et c., et éventuellement produit à l’étape d. est un altPEP (ou non). C’est d’ailleurs ce que permet l’alternative dite de comparaison phénotypique mise en œuvre à l’étape e. Par exemple, si le peptide déterminé sur l’ARNm d’une protéine impliquée dans la taille de la tige d’une plante provoque une augmentation, ou une diminution, de la taille de la tige d’une plante traitée avec ce dernier par rapport à une plante non traitée, cela signifie que ledit peptide est un altPEP capable de moduler l’accumulation de ladite protéine dans la taille de la tige. Dans l’invention, le terme « plante » fait référence de manière générale : à un ensemble de cellules végétales organisé en tout ou partie d’une plante quel que soit son stade de développement (y compris la plante sous forme de graine ou de jeune pousse) ; à un ou plusieurs organes de la plante (comme par exemple les feuilles, les racines, la tige, les fleurs) ; à une ou plusieurs cellules de la plante ; ou encore à un amas de cellules de la plante {e.g. un cal). The inventors have surprisingly noted that the use of altPEPs makes it possible to modify the phenotypes of a plant visible on a macroscopic scale. It is therefore entirely possible to use the latter to affirm (or refute) that the peptide determined in steps a., b. and c., and optionally produced in step d. is an altPEP (or not). This is also what the so-called phenotypic comparison alternative implemented in step e allows. For example, if the peptide determined on the mRNA of a protein involved in the size of the stem of a plant causes an increase, or a decrease, in the size of the stem of a plant treated with the latter compared to an untreated plant, this means that said peptide is an altPEP capable of modulating the accumulation of said protein in the size of the stem. In the invention, the term "plant" refers generally: to a set of plant cells organized in all or part of a plant whatever its stage of development (including the plant in the form of seed or young grows); to one or more organs of the plant (such as leaves, roots, stem, flowers); to one or more cells of the plant; or even a mass of plant cells {eg a callus).
Dans l’invention, le terme « phénotype » désigne de manière non limitative les caractères visibles à l’échelle macroscopique tel que le nombre de racines latérales, le nombre de feuilles, la taille de la tige, la durée de floraison et la résistance au stress. Dans un mode de réalisation, l’invention concerne par conséquent le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est impliquée dans au moins un phénotype de plante choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In the invention, the term "phenotype" designates in a non-limiting manner the characters visible on a macroscopic scale such as the number of lateral roots, the number of leaves, the size of the stem, the duration of flowering and the resistance to stress. In one embodiment, the invention therefore relates to the method for preparing and determining an altPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Selon l’invention, une protéine est « impliquée dans un phénotype » si une modification de l’accumulation de celle-ci est associée à une modification dudit phénotype. En d’autres termes, une protéine est impliquée dans un phénotype si elle intervient dans le(s) caractère(s) correspondant audit phénotype. According to the invention, a protein is “involved in a phenotype” if a modification of the accumulation thereof is associated with a modification of said phenotype. In other words, a protein is involved in a phenotype if it intervenes in the character(s) corresponding to said phenotype.
Au vu de ce qui précède, on comprend qu’un objet de l’invention est le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel le phénotype observé à l’étape e. est choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In view of the above, it is understood that an object of the invention is the process for preparing and determining an altPEP as described above, in which the phenotype observed in step e. is chosen from: the size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, ledit altPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés ; étant capable de moduler l’accumulation d’une protéine dans une cellule végétale ; et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, et dans lequel ladite cellule végétale (/.e. celle dans laquelle on souhaite moduler l’accumulation d’une protéine) appartient à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, said altPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, and in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) belongs to a chosen plant species among: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel lesdites cellules végétales ou lesdites plantes utilisées à l’étape e. appartiennent à : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said plant cells or said plants used in step e. belong to: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora , Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite cellule végétale (/.e. celle dans laquelle on souhaite moduler l’accumulation d’une protéine) est une cellule d’une algue. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) is a cell of an algae.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel lesdites cellules végétales ou lesdites plantes utilisées à l’étape e appartiennent à une algue. In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said plant cells or said plants used in step e belong to an alga.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi : Aae15 (Acyl-activating enzyme 15), Aae16 (AMP-dependent synthetase and ligase family protein), Abcg11 (White-brown complex-like protein), Abdcg34 (ABC transporter G family member 34), Acc1 (Acetyl-CoA Carboxylase), Agb1 (GTP binding protein beta 1), Als (Acetolactate synthase (chloroplastic)), Anac076 (NAC domain-containing protein 76), Apg9 (Autophagy 9), Arlbl (GTP-binding protein 1), Arr1 (Two-component response regulator ARR1), Arr5 (Two-component response regulator ARR5), Arr6 (Two- component response regulator ARR6), At59 (Pedate lyase family protein), Bak1 (Brassinosteroid insensitive 1 -associated receptor kinase 1), Bccpl (Acetyl-CoA Carboxylase (chloroplastic) subunit 1), Bccp2 (Acetyl-CoA Carboxylase (chloroplastic) subunit 2), Bril (Brassinosteroid insensitive 1), Bzo2h3 (bZIP transcription factor family protein), Cesa6 (Cellulose synthase A catalytic subunit 6), Cipk3 (CBL-intera ing protein kinase 3), Cks1 (Cyclin-dependent kinases regulatory subunit 1), Cobl8 (COBRA-like protein 8 precursor), Coil (Coronatine-insensitive protein 1), Cpk3 (Calcium-dependent protein kinase 3), Crk34 (Cysteine-rich receptor-like protein kinase 34), Cyp705a18 (Cytochrome P450, family 705, subfamily A, polypeptide 18), Cyp71b26 (Cytochrome P450, family 71, subfamily B, polypeptide 26), Cyp78a8 (Cytochrome P450, family 78, subfamily A, polypeptide 8), Cyp97b3 (Cytochrome P450, family 97, subfamily B, polypeptide 3), Dell (Endoribonuclease Dicer homolog 1), Dur3 (Urea-proton symporter DUR3), Ein2 (Ethylene-insensitive protein 2), Emb 175 (Pentatricopeptide repeat-containing protein), Emb2726 (Elongation factor Ts family protein), Emb9 (Di hydrofolate synthetase), Epsps (5-enolpyruvylshikimate-3-phosphate (chloroplastic)), Fnr1 (Ferredoxin-NADP[+]-oxidoreductase 1), Fve (Transducin family protein / WD-40 repeat family protein), Ga2ox7 (Gibberellin 2-beta-dioxygenase 7), Gape (Glyceraldehyde-3-phosphate dehydrogenase), Gcn2 (ABC transporter family protein), Gdi2 (Guanosine nucleotide diphosphate dissociation inhibitor 2), Gln2 (Glutamine synthetase (chloroplastic)), Gsl3 (Callose synthase 2), Hag5 (Histone acetyltransferase of the MYST family 2), Hda18 (Histone deacetylase 18), Hexol (Beta-hexosaminidase 1), Hppd (4- hydroxyphenyl-pyruvate-dioxygenase), Hsl1 (B3 domain-containing transcription repressor VAL2), Iaa31 (lndole-3-acetic acid inducible 31), Iqd28 (IQ-domain 28), Jac1 (J-domain protein required for chloroplast accumulation response 1), Jar1 (Jasmonoyl-L-amino acid synthetase), Kp1 (Kinesin-like protein 1), Lrx2 (Leucine-rich repeat/extensin 2), Mapkkk3 (Mitogen-activated protein kinase kinase kinase 3), Mapkkk5 (Mitogen-activated protein kinase kinase kinase 5), Mfp2 (Multifunctional protein 2), Mrb1 (Transmembrane protein, putative (DUF3537)), Nsp1 (Nodulation signaling pathway 1), Pds (Phytoene desaturase (chloroplastic)), Pen3 (Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein- tyrosine-phosphatase), Phyb (Phytochrome B), Pif3 (Phytochrome interacting factor 3), Pizza (Brassinosteroid-related acyltransferase 1), Ppoxl (Protoporphyrinogen oxidase (chloroplastic) 1), Ppox2 (Protoporphyrinogen oxidase (chloroplastic) 2), Prp39 (Tetratricopeptide repeat (TPR)-like superfamily protein), PsbA (Photosystem II D1 protein), Pskrl (Phytosulfokin receptor 1), Rd21 (Granulin repeat cysteine protease family protein), Ringl (RING/U-box superfamily protein), Rosi (DNA glycosylase/AP lyase ROS1), Rpt4a (26S proteasome regulatory subunit 10B homolog A), Sfr6 (Mediator of RNA polymerase II transcription subunit 16), Shr (Protein SHORT-ROOT), Shy2 (Auxin-responsive protein IAA3), Ski (EIN2-like protein, nramp transporter), Sps1 (Sucrose phosphate synthase 2F), Spt (Transcription factor SPATULA), Stn8 (Serine/threonine-protein kinase), Tap46 (PP2A regulatory subunit TAP46), Topp6 (Serine/threonine-protein phosphatase PP1 isozyme 7), TubB6 (Tubulin), TubB8 (Tubulin), Ubala (RNA-binding (RRM/RBD/RNP motifs) family protein), Vim3 (E3 ubiquitin-protein ligase), Sgr1 (Magnesium dechelatase), Abi5 (Abscisic acid (ABA)-insensitive 5), Hsp101 (Heat shock protein 101), Rh10 (ATP-dependent RNA helicase) et Wus (WUSCHEL). In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene chosen from: Aae15 (Acyl-activating enzyme 15), Aae16 (AMP -dependent synthetase and ligase family protein), Abcg11 (White-brown complex-like protein), Abdcg34 (ABC transporter G family member 34), Acc1 (Acetyl-CoA Carboxylase), Agb1 (GTP binding protein beta 1), Als (Acetolactate synthase (chloroplastic)), Anac076 (NAC domain-containing protein 76), Apg9 (Autophagy 9), Arlbl (GTP-binding protein 1), Arr1 (Two-component response regulator ARR1), Arr5 (Two-component response regulator ARR5) , Arr6 (Two-component response regulator ARR6), At59 (Pedate lyase family protein), Bak1 (Brassinosteroid insensitive 1 -associated receptor kinase 1), Bccpl (Acetyl-CoA Carboxylase (chloroplastic) subunit 1), Bccp2 (Acetyl-CoA Carboxylase (chloroplastic) subunit 2), Bril (Brassinosteroid insensitive 1), Bzo2h3 (bZIP transcription factor family protein), Cesa6 (Cellulose synthase A catalytic subunit 6), Cipk3 (CBL-intera ing protein kinase 3), Cks1 (Cyclin-dependent kinases regulatory subunit 1), Cobl8 (COBRA-like protein 8 precursor), Coil (Coronatine-insensitive protein 1), Cpk3 (Calcium-dependent protein kinase 3), Crk34 (Cysteine-rich receptor-like protein kinase 34), Cyp705a18 (Cytochrome P450, family 705, subfamily A, polypeptide 18), Cyp71b26 (Cytochrome P450, family 71, subfamily B, polypeptide 26), Cyp78a8 (Cytochrome P450, family 78, subfamily A, polypeptide 8), Cyp97b3 (Cytochrome P450, family 97, subfamily B, polypeptide 3), Dell (Endoribonuclease Dicer homolog 1), Dur3 (Urea-proton symporter DUR3) , Ein2 (Ethylene-insensitive protein 2), Emb 175 (Pentatricopeptide repeat-containing protein), Emb2726 (Elongation factor Ts family protein), Emb9 (Di hydrofolate synthetase), Epsps (5-enolpyruvylshikimate-3-phosphate (chloroplastic)), Fnr1 (Ferredoxin-NADP[+]-oxidoreductase 1), Fve (Transducin family protein / WD-40 repeat family protein), Ga2ox7 (Gibberellin 2-beta-dioxygenase 7), Gape (Glyceraldehyde-3-phosphate dehydrogenase), Gcn2 ( ABC transporter family protein), Gdi2 (Guanosine nucleotide diphosphate dissociation inhibitor 2), Gln2 (Glutamine synthetase (chloroplastic)), Gsl3 (Callose synthase 2), Hag5 (Histone acetyltransferase of the MYST family 2), Hda18 (Histone deacetylase 18), Hexol (Beta-hexosaminidase 1), Hppd (4-hydroxyphenyl-pyruvate-dioxygenase), Hsl1 (B3 domain-containing transcription repressor VAL2), Iaa31 (lndole-3-acetic acid inducible 31), Iqd28 (IQ-domain 28), Jac1 (J-domain protein required for chloroplast accumulation response 1), Jar1 (Jasmonoyl-L-amino acid synthetase), Kp1 (Kinesin-like protein 1), Lrx2 (Leucine-rich repeat/extensin 2), Mapkkk3 (Mitogen-activated protein kinase kinase kinase kinase 3), Mapkkk5 (Mitogen-activated protein kinase kinase kinase 5), Mfp2 (Multifunctional protein 2), Mrb1 (Transmembrane protein, putative (DUF3537)), Nsp1 (Nodulation signaling pathway 1), Pds (Phytoene desaturase ( chloroplastic)), Pen3 (Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein-tyrosine-phosphatase), Phyb (Phytochrome B), Pif3 (Phytochrome interacting factor 3), Pizza (Brassinosteroid-related acyltransferase 1), Ppoxl ( Protoporphyrinogen oxidase (chloroplastic) 1), Ppox2 (Protoporphyrinogen oxidase (chloroplastic) 2), Prp39 (Tetratricopeptide repeat (TPR)-like superfamily protein), PsbA (Photosystem II D1 protein), Pskrl (Phytosulfokin receptor 1), Rd21 (Granulin repeat cysteine protease family protein), Ringl (RING/U-box superfamily protein), Rosi (DNA glycosylase/AP lyase ROS1), Rpt4a (26S proteasome regulatory subunit 10B homolog A), Sfr6 (Mediator of RNA polymerase II transcription subunit 16), Shr (SHORT-ROOT protein), Shy2 (Auxin-responsive protein IAA3), Ski (EIN2-like protein, nramp transporter), Sps1 (Sucrose phosphate synthase 2F), Spt (Transcription factor SPATULA), Stn8 (Serine/threonine-protein kinase), Tap46 (PP2A regulatory subunit TAP46), Topp6 (Serine/threonine-protein phosphatase PP1 isozyme 7), TubB6 (Tubulin), TubB8 (Tubulin), Ubala (RNA-binding (RRM/RBD/RNP motifs) family protein) , Vim3 (E3 ubiquitin-protein ligase), Sgr1 (Magnesium dechelatase), Abi5 (Abscisic acid (ABA)-insensitive 5), Hsp101 (Heat shock protein 101), Rh10 (ATP-dependent RNA helicase) and Wus (WUSCHEL).
En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi : Cpk3, Dell et Nsp1. En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Cpk3. En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Dell. En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Nsp1. In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene chosen from: Cpk3, Dell and Nsp1. In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by the Cpk3 gene. In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by the Dell gene. In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by the Nsp1 gene.
Les gènes Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 et l/l/us font références aux protéines indiquées entre parenthèses. Bien entendu, l’invention concerne également des gènes homologues et/ou similaires susceptibles de porter des dénominations différentes. Par exemple, chez A. thaliana le gène Gsl3 codant pour la callose synthase 2 s’appelle également Cals2. The genes Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi , Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 and l/l/us refer to proteins indicated in parentheses. Of course, the invention also relates to homologous and/or similar genes likely to bear different names. For example, in A. thaliana the Gsl3 gene encoding callose synthase 2 is also called Cals2.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NO : 1 (ORF de la protéine Aae15, A. thaliana), SEQ ID NO : 2 (ORF de la protéine Aae16, A. thaliana), SEQ ID NO : 3 (ORF de la protéine abcg11, A. thaliana), SEQ ID NO : 4 (ORF de la protéine Abdcg34, A. thaliana), SEQ ID NO : 5 (ORF de la protéine Acc1, A. thaliana), SEQ ID NO : 6 (ORF de la protéine Agb1 , A. thaliana), SEQ ID NO : 7 (ORF de la protéine Als, A. thaliana), SEQ ID NO : 8 (ORF de la protéine Anac076, A. thaliana), SEQ ID NO : 9 (ORF de la protéine Apg9, A. thaliana), SEQ ID NO : 10 (ORF de la protéine Arlbl, A. thaliana), SEQ ID NO : 11 (ORF de la protéine Arr1, A. thaliana), SEQ ID NO : 12 (ORF de la protéine Arr5, A. thaliana), SEQ ID NO : 13 (ORF de la protéine Arr6, A. thaliana), SEQ ID NO : 14 (ORF de la protéine At59, A. thaliana), SEQ ID NO : 15 (ORF de la protéine Bak1, A. thaliana), SEQ ID NO : 16 (ORF de la protéine Bccpl , A. thaliana), SEQ ID NO : 17 (ORF de la protéine Bccp2, A. thaliana), SEQ ID NO : 18 (ORF de la protéine Bri1 , A. thaliana), SEQ ID NO : 19 (ORF de la protéine Bzo2h3, A. thaliana), SEQ ID NO : 20 (ORF de la protéine Cesa6, A. thaliana), SEQ ID NO : 21 (ORF de la protéine Cipk3, A. thaliana), SEQ ID NO : 22 (ORF de la protéine Cks1, A thaliana), SEQ ID NO : 23 (ORF de la protéine Cobl8, A thaliana), SEQ ID NO : 24 (ORF de la protéine Coi1, A. thaliana), SEQ ID NO : 25 (ORF de la protéine Coil, A. thaliana), SEQ ID NO : 26 (ORF de la protéine Cpk3, A. thaliana), SEQ ID NO : 27 (ORF de la protéine Cpk3, A. hypochondriacus), SEQ ID NO : 28 (ORF de la protéine Cpk3, B. distachyori), SEQ ID NO : 29 (ORF de la protéine Cpk3, B. distachyori), SEQ ID NO : 30 (ORF de la protéine Cpk3, G. max), SEQ ID NO : 31 (ORF de la protéine Cpk3, G. max), SEQ ID NO : 32 (ORF de la protéine Cpk3, G. max), SEQ ID NO : 33 (ORF de la protéine Cpk3, G. max), SEQ ID NO : 34 (ORF de la protéine Cpk3, O. sativa), SEQ ID NO : 35 (ORF de la protéine Cpk3, O. sativa), SEQ ID NO : 36 (ORF de la protéine Cpk3, S. lycopersicum), SEQ ID NO : 37 (ORF de la protéine Cpk3, Z. mays), SEQ ID NO : 38 (ORF de la protéine Cpk3, Z. mays), SEQ ID NO : 39 (ORF de la protéine Cpk3, Z. mays), SEQ ID NO : 40 (ORF de la protéine Cpk3, B. rapa), SEQ ID NO : 41 (ORF de la protéine Cpk3, B. rapa), SEQ ID NO : 42 (ORF de la protéine Cpk3, H. vulgare), SEQ ID NO : 43 (ORF de la protéine Cpk3, H. vulgare), SEQ ID NO : 44 (ORF de la protéine Cpk3, S. tuberosum), SEQ ID NO : 45 (ORF de la protéine Cpk3, A. palmeri), SEQ ID NO : 46 (ORF de la protéine Cpk3, M. truncatula), SEQ ID NO : 47 (ORF de la protéine Cpk3, M. truncatula), SEQ ID NO : 48 (ORF de la protéine Cpk3, T. aestivum), SEQ ID NO : 49 (ORF de la protéine Cpk3, T. aestivum), SEQ ID NO : 50 (ORF de la protéine Cpk3, T. aestivum), SEQ ID NO : 51 (ORF de la protéine Cpk3, T. aestivum), SEQ ID NO : 52 (ORF de la protéine Cpk3, L. perenne), SEQ ID NO : 53 (ORF de la protéine Cpk3, L. perenne), SEQ ID NO : 54 (ORF de la protéine Cpk3, L. perenne), SEQ ID NO : 55 (ORF de la protéine Cpk3, L. perenne), SEQ ID NO : 56 (ORF de la protéine Crk34, A. thaliana), SEQ ID NO : 57 (ORF de la protéine Cyp705a18, A. thaliana), SEQ ID NO : 58 (ORF de la protéine Cyp71b26, A. thaliana), SEQ ID NO : 59 (ORF de la protéine Cyp78a8, A. thaliana), SEQ ID NO : 60 (ORF de la protéine Cyp97b3, A. thaliana), SEQ ID NO : 61 (ORF de la protéine Dell, A. thaliana), SEQ ID NO : 62 (ORF de la protéine Dell, A. thaliana), SEQ ID NO : 63 (ORF de la protéine Dell , A. hypochondriacus), SEQ ID NO : 64 (ORF de la protéine Dell, B. distachyon), SEQ ID NO : 65 (ORF de la protéine Dell, G. max), SEQ ID NO : 66 (ORF de la protéine Dell, G. max), SEQ ID NO : 67 (ORF de la protéine Dell, O. sativa), SEQ ID NO : 68 (ORF de la protéine Dell , S. lycopersicum), SEQ ID NO : 69 (ORF de la protéine Dell , Z. mays), SEQ ID NO : 70 (ORF de la protéine Dell , B. rapa), SEQ ID NO : 71 (ORF de la protéine Dell, H. vulgare), SEQ ID NO : 72 (ORF de la protéine Dell, S. tuberosum), SEQ ID NO : 73 (ORF de la protéine Dell, M. truncatula), SEQ ID NO : 74 (ORF de la protéine Dell, T. aestivum), SEQ ID NO : 75 (ORF de la protéine Dell, T. aestivum), SEQ ID NO : 76 (ORF de la protéine Dell, T. aestivum), SEQ ID NO : 77 (ORF de la protéine Dell, T. aestivum), SEQ ID NO : 78 (ORF de la protéine Dell , L. perenne), SEQ ID NO : 79 (ORF de la protéine Dell , L. perenne), SEQ ID NO : 80 (ORF de la protéine Dur3, A thaliana), SEQ ID NO : 81 (ORF de la protéine Ein2, Æ thaliana), SEQ ID NO : 82 (ORF de la protéine Emb175, A. thaliana), SEQ ID NO : 83 (ORF de la protéine Emb2726, A. thaliana), SEQ ID NO : 84 (ORF de la protéine Emb9, A. thaliana), SEQ ID NO : 85 (ORF de la protéine Epsps, A. thaliana), SEQ ID NO : 86 (ORF de la protéine Fnr1 , A. thaliana), SEQ ID NO : 87 (ORF de la protéine Fve, A. thaliana), SEQ ID NO : 88 (ORF de la protéine Ga2ox7, A. thaliana), SEQ ID NO : 89 (ORF de la protéine Gape, N. benthamiana), SEQ ID NO : 90 (ORF de la protéine Gcn2, A. thaliana), SEQ ID NO : 91 (ORF de la protéine Gdi2, A. thaliana), SEQ ID NO : 92 (ORF de la protéine Gln2, A. thaliana), SEQ ID NO : 93 (ORF de la protéine Gsl3, A. thaliana), SEQ ID NO : 94 (ORF de la protéine Hag5, A. thaliana), SEQ ID NO : 95 (ORF de la protéine Hda18, A. thaliana), SEQ ID NO : 96 (ORF de la protéine Hexol , A. thaliana), SEQ ID NO : 97 (ORF de la protéine Hppd, A. thaliana), SEQ ID NO : 98 (ORF de la protéine Hsl1, A. thaliana), SEQ ID NO : 99 (ORF de la protéine Iaa31, A. thaliana), SEQ ID NO : 100 (ORF de la protéine Iqd28, A. thaliana), SEQ ID NO : 101 (ORF de la protéine Jac1 , A. thaliana), SEQ ID NO : 102 (ORF de la protéine Jar1 , A. thaliana), SEQ ID NO : 103 (ORF de la protéine Kp1 , A. thaliana), SEQ ID NO : 104 (ORF de la protéine Lrx2, A. thaliana), SEQ ID NO : 105 (ORF de la protéine Mapkkk3, A. thaliana), SEQ ID NO : 106 (ORF de la protéine Mapkkk5, A. thaliana), SEQ ID NO : 107 (ORF de la protéine Mfp2, A. thaliana), SEQ ID NO : 108 (ORF de la protéine Mrb1, A. thaliana), SEQ ID NO : 109 (ORF de la protéine Nsp1, M. truncatula), SEQ ID NO : 110 (ORF de la protéine Nsp1, A. thaliana), SEQ ID NO : 111 (ORF de la protéine Nsp1 , B. distachyon), SEQ ID NO : 112 (ORF de la protéine Nsp1, G. max), SEQ ID NO : 113 (ORF de la protéine Nsp1, G. max), SEQ ID NO : 114 (ORF de la protéine Nsp1, O. sativa), SEQ ID NO : 115 (ORF de la protéine Nsp1 , S. lycopersicum), SEQ ID NO : 116 (ORF de la protéine Nsp1, S. lycopersicum), SEQ ID NO : 117 (ORF de la protéine Nsp1, Z. mays), SEQ ID NO : 118 (ORF de la protéine Nsp1, Z. mays), SEQ ID NO : 119 (ORF de la protéine Nsp1, Z. mays), SEQ ID NO : 120 (ORF de la protéine Nsp1 , Z. mays), SEQ ID NO : 121 (ORF de la protéine Nsp1 , B. rapa), SEQ ID NO : 122 (ORF de la protéine Nsp1 , H. vulgare), SEQ ID NO : 123 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 124 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 125 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 126 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 127 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 128 (ORF de la protéine Nsp1, H. vulgare), SEQ ID NO : 129 (ORF de la protéine Nsp1 , S. tuberosum), SEQ ID NO : 130 (ORF de la protéine Nsp1, S. tuberosum), SEQ ID NO : 131 (ORF de la protéine Nsp1, T. aestivum), SEQ ID NO : 132 (ORF de la protéine Nsp1 , T. aestivum), SEQ ID NO : 133 (ORF de la protéine Nsp1 , L. perenne), SEQ ID NO : 134 (ORF de la protéine Nsp1, L. perenne), SEQ ID NO : 135 (ORF de la protéine Pds, A. thaliana), SEQ ID NO : 136 (ORF de la protéine Pen3, A. thaliana), SEQ ID NO : 137 (ORF de la protéine Phyb, A. thaliana), SEQ ID NO : 138 (ORF de la protéine Pif3, A. thaliana), SEQ ID NO : 139 (ORF de la protéine Pizza, A thaliana), SEQ ID NO : 140 (ORF de la protéine Ppoxl, A thaliana), SEQ ID NO : 141 (ORF de la protéine Ppox2, A. thaliana), SEQ ID NO : 142 (ORF de la protéine Prp39, A. thaliana), SEQ ID NO : 143 (ORF de la protéine PsbA, A. thaliana), SEQ ID NO : 144 (ORF de la protéine Pskrl, A. thaliana), SEQ ID NO : 145 (ORF de la protéine Rd21, A. thaliana), SEQ ID NO : 146 (ORF de la protéine Ringl, A. thaliana), SEQ ID NO : 147 (ORF de la protéine Rosi , A. thaliana), SEQ ID NO : 148 (ORF de la protéine Rpt4a, A. thaliana), SEQ ID NO : 149 (ORF de la protéine Sfr6, A. thaliana), SEQ ID NO : 150 (ORF de la protéine Shr, A. thaliana), SEQ ID NO : 151 (ORF de la protéine Shy2, A. thaliana), SEQ ID NO : 152 (ORF de la protéine Ski, M. truncatula), SEQ ID NO : 153 (ORF de la protéine Sps1 , A. thaliana), SEQ ID NO : 154 (ORF de la protéine Spt, A. thaliana), SEQ ID NO : 155 (ORF de la protéine Stn8, A. thaliana), SEQ ID NO : 156 (ORF de la protéine Tap46, A. thaliana), SEQ ID NO : 157 (ORF de la protéine Topp6, A. thaliana), SEQ ID NO : 158 (ORF de la protéine TubB6, A. thaliana), SEQ ID NO : 159 (ORF de la protéine TubB8, A. thaliana), SEQ ID NO : 160 (ORF de la protéine llbala, A. thaliana), SEQ ID NO : 161 (ORF de la protéine Vim3, A. thaliana), SEQ ID NO : 381 (ORF de la protéine Sgr1 , A. thaliana), SEQ ID NO : 382 (ORF de la protéine Abi5, A. thaliana), SEQ ID NO : 383 (ORF de la protéine Hsp101, A. thaliana), SEQ ID NO : 384 (ORF de la protéine Rh10, M. truncatula) et SEQ ID NO : 385 (ORF de la protéine Wus, A. thaliana). In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity. , preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NO: 1 (ORF of the Aae15 protein, A. thaliana), SEQ ID NO: 2 (ORF of the Aae16 protein, A. thaliana), SEQ ID NO: 3 (ORF of the abcg11 protein, A. thaliana), SEQ ID NO: 4 (ORF of the Abdcg34 protein, A. thaliana), SEQ ID NO: 5 (ORF of the Acc1 protein, A . thaliana), SEQ ID NO: 6 (ORF of the Agb1 protein, A. thaliana), SEQ ID NO: 7 (ORF of the Als protein, A. thaliana), SEQ ID NO: 8 (ORF of the Anac076 protein, A. thaliana), SEQ ID NO: 9 (ORF of the Apg9 protein, A. thaliana), SEQ ID NO: 10 (ORF of the Arlbl protein, A. thaliana), SEQ ID NO: 11 (ORF of the Arr1 protein , A. thaliana), SEQ ID NO: 12 (ORF of the Arr5 protein, A. thaliana), SEQ ID NO: 13 (ORF of the Arr6 protein, A. thaliana), SEQ ID NO: 14 (ORF of the protein At59, A. thaliana), SEQ ID NO: 15 (ORF of the Bak1 protein, A. thaliana), SEQ ID NO: 16 (ORF of the Bccpl protein, A. thaliana), SEQ ID NO: 17 (ORF of the Bccp2 protein, A. thaliana), SEQ ID NO: 18 (ORF of the Bri1 protein, A. thaliana), SEQ ID NO: 19 (ORF of the Bzo2h3 protein, A. thaliana), SEQ ID NO: 20 (ORF of the Cesa6 protein, A. thaliana), SEQ ID NO: 21 (ORF of the Cipk3 protein, A. thaliana), SEQ ID NO: 22 (ORF of the Cks1 protein, A thaliana), SEQ ID NO: 23 (ORF of the Cobl8 protein, A thaliana), SEQ ID NO: 24 (ORF of the Coi1 protein, A. thaliana), SEQ ID NO: 25 ( ORF of the Coil protein, A. thaliana), SEQ ID NO: 26 (ORF of the Cpk3 protein, A. thaliana), SEQ ID NO: 27 (ORF of the Cpk3 protein, A. hypochondriacus), SEQ ID NO: 28 (ORF of the Cpk3 protein, B. distachyori), SEQ ID NO: 29 (ORF of the Cpk3 protein, B. distachyori), SEQ ID NO: 30 (ORF of the Cpk3 protein, G. max), SEQ ID NO: 31 (ORF of the Cpk3 protein, G. max), SEQ ID NO: 32 (ORF of the Cpk3 protein, G. max), SEQ ID NO: 33 (ORF of the Cpk3 protein, G. max), SEQ ID NO : 34 (ORF of the Cpk3 protein, O. sativa), SEQ ID NO: 35 (ORF of the Cpk3 protein, O. sativa), SEQ ID NO: 36 (ORF of the Cpk3 protein, S. lycopersicum), SEQ ID NO: 37 (ORF of the Cpk3 protein, Z. mays), SEQ ID NO: 38 (ORF of the Cpk3 protein, Z. mays), SEQ ID NO: 39 (ORF of the Cpk3 protein, Z. mays), SEQ ID NO: 40 (ORF of the Cpk3 protein, B. rapa), SEQ ID NO: 41 (ORF of the Cpk3 protein, B. rapa), SEQ ID NO: 42 (ORF of the Cpk3 protein, H. vulgare), SEQ ID NO: 43 (ORF of the Cpk3 protein, H. vulgare), SEQ ID NO: 44 (ORF of the Cpk3 protein, S. tuberosum), SEQ ID NO: 45 (ORF of the Cpk3 protein, A. palmeri) , SEQ ID NO: 46 (ORF of the Cpk3 protein, M. truncatula), SEQ ID NO: 47 (ORF of the Cpk3 protein, M. truncatula), SEQ ID NO: 48 (ORF of the Cpk3 protein, T. aestivum ), SEQ ID NO: 49 (ORF of the Cpk3 protein, T. aestivum), SEQ ID NO: 50 (ORF of the Cpk3 protein, T. aestivum), SEQ ID NO: 51 (ORF of the Cpk3 protein, T. aestivum), SEQ ID NO: 52 (ORF of the Cpk3 protein, L. perenne), SEQ ID NO: 53 (ORF of the Cpk3 protein, L. perenne), SEQ ID NO: 54 (ORF of the Cpk3 protein, L . perenne), SEQ ID NO: 55 (ORF of the Cpk3 protein, L. perenne), SEQ ID NO: 56 (ORF of the Crk34 protein, A. thaliana), SEQ ID NO: 57 (ORF of the Cyp705a18 protein, A. thaliana), SEQ ID NO: 58 (ORF of the Cyp71b26 protein, A. thaliana), SEQ ID NO: 59 (ORF of the Cyp78a8 protein, A. thaliana), SEQ ID NO: 60 (ORF of the Cyp97b3 protein , A. thaliana), SEQ ID NO: 61 (ORF of the Dell protein, A. thaliana), SEQ ID NO: 62 (ORF of the Dell protein, A. thaliana), SEQ ID NO: 63 (ORF of the protein Dell, A. hypochondriacus), SEQ ID NO: 64 (ORF of the Dell protein, B. distachyon), SEQ ID NO: 65 (ORF of the Dell protein, G. max), SEQ ID NO: 66 (ORF of the Dell protein, G. max), SEQ ID NO: 67 (ORF of the Dell protein, O. sativa), SEQ ID NO: 68 (ORF of the Dell protein, S. lycopersicum), SEQ ID NO: 69 (ORF of the Dell protein, Z. mays), SEQ ID NO: 70 (ORF of the Dell protein, B. rapa), SEQ ID NO: 71 (ORF of the Dell protein, H. vulgare), SEQ ID NO: 72 (ORF of the Dell protein, S. tuberosum), SEQ ID NO: 73 (ORF of the Dell protein, M. truncatula), SEQ ID NO: 74 (ORF of the Dell protein, T. aestivum), SEQ ID NO: 75 ( ORF of the Dell protein, T. aestivum), SEQ ID NO: 76 (ORF of the Dell protein, T. aestivum), SEQ ID NO: 77 (ORF of the Dell protein, T. aestivum), SEQ ID NO: 78 (ORF of the Dell protein, L. perenne), SEQ ID NO: 79 (ORF of the Dell protein, L. perenne), SEQ ID NO: 80 (ORF of the Dur3 protein, A thaliana), SEQ ID NO: 81 (ORF of the Ein2 protein, A. thaliana), SEQ ID NO: 82 (ORF of the Emb175 protein, A. thaliana), SEQ ID NO: 83 (ORF of the Emb2726 protein, A. thaliana), SEQ ID NO: 84 (ORF of the Emb9 protein, A. thaliana), SEQ ID NO: 85 (ORF of the Epsps protein, A. thaliana), SEQ ID NO: 86 (ORF of the Fnr1 protein, A. thaliana) , SEQ ID NO: 87 (ORF of the Fve protein, A. thaliana), SEQ ID NO: 88 (ORF of the Ga2ox7 protein, A. thaliana), SEQ ID NO: 89 (ORF of the Gape protein, N. benthamiana ), SEQ ID NO: 90 (ORF of the Gcn2 protein, A. thaliana), SEQ ID NO: 91 (ORF of the Gdi2 protein, A. thaliana), SEQ ID NO: 92 (ORF of the Gln2 protein, A. thaliana), SEQ ID NO: 93 (ORF of the Gsl3 protein, A. thaliana), SEQ ID NO: 94 (ORF of the Hag5 protein, A. thaliana), SEQ ID NO: 95 (ORF of the Hda18 protein, A . thaliana), SEQ ID NO: 96 (ORF of the Hexol protein, A. thaliana), SEQ ID NO: 97 (ORF of the Hppd protein, A. thaliana), SEQ ID NO: 98 (ORF of the Hsl1 protein, A. thaliana), SEQ ID NO: 99 (ORF of the Iaa31 protein, A. thaliana), SEQ ID NO: 100 (ORF of the Iqd28 protein, A. thaliana), SEQ ID NO: 101 (ORF of the Jac1 protein , A. thaliana), SEQ ID NO: 102 (ORF of the Jar1 protein, A. thaliana), SEQ ID NO: 103 (ORF of the Kp1 protein, A. thaliana), SEQ ID NO: 104 (ORF of the protein Lrx2, A. thaliana), SEQ ID NO: 105 (ORF of the Mapkkk3 protein, A. thaliana), SEQ ID NO: 106 (ORF of the Mapkkk5 protein, A. thaliana), SEQ ID NO: 107 (ORF of the Mfp2 protein, A. thaliana), SEQ ID NO: 108 (ORF of the Mrb1 protein, A. thaliana), SEQ ID NO: 109 (ORF of the Nsp1 protein, M. truncatula), SEQ ID NO: 110 (ORF of the Nsp1 protein, A. thaliana), SEQ ID NO: 111 (ORF of the Nsp1 protein, B. distachyon), SEQ ID NO: 112 (ORF of the Nsp1 protein, G. max), SEQ ID NO: 113 (ORF of the Nsp1 protein, G. max), SEQ ID NO: 114 (ORF of the Nsp1 protein, O. sativa), SEQ ID NO: 115 (ORF of the Nsp1 protein, S. lycopersicum), SEQ ID NO: 116 ( ORF of the Nsp1 protein, S. lycopersicum), SEQ ID NO: 117 (ORF of the Nsp1 protein, Z. mays), SEQ ID NO: 118 (ORF of the Nsp1 protein, Z. mays), SEQ ID NO: 119 (ORF of the Nsp1 protein, Z. mays), SEQ ID NO: 120 (ORF of the Nsp1 protein, Z. mays), SEQ ID NO: 121 (ORF of the Nsp1 protein, B. rapa), SEQ ID NO: 122 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 123 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 124 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO : 125 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 126 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 127 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 128 (ORF of the Nsp1 protein, H. vulgare), SEQ ID NO: 129 (ORF of the Nsp1 protein, S. tuberosum), SEQ ID NO: 130 (ORF of the Nsp1 protein, S. tuberosum), SEQ ID NO: 131 (ORF of the Nsp1 protein, T. aestivum), SEQ ID NO: 132 (ORF of the Nsp1 protein, T. aestivum), SEQ ID NO: 133 (ORF of the Nsp1 protein, L. perenne), SEQ ID NO: 134 (ORF of the Nsp1 protein, L. perenne), SEQ ID NO: 135 (ORF of the Pds protein, A. thaliana), SEQ ID NO: 136 (ORF of the Pen3 protein, A. thaliana) , SEQ ID NO: 137 (ORF of the Phyb protein, A. thaliana), SEQ ID NO: 138 (ORF of the Pif3 protein, A. thaliana), SEQ ID NO: 139 (ORF of the Pizza protein, A thaliana), SEQ ID NO: 140 (ORF of the Ppoxl protein, A thaliana), SEQ ID NO: 141 (ORF of the Ppox2 protein, A. thaliana), SEQ ID NO: 142 ( ORF of the Prp39 protein, A. thaliana), SEQ ID NO: 143 (ORF of the PsbA protein, A. thaliana), SEQ ID NO: 144 (ORF of the Pskrl protein, A. thaliana), SEQ ID NO: 145 (ORF of the Rd21 protein, A. thaliana), SEQ ID NO: 146 (ORF of the Ringl protein, A. thaliana), SEQ ID NO: 147 (ORF of the Rosi protein, A. thaliana), SEQ ID NO: 148 (ORF of the Rpt4a protein, A. thaliana), SEQ ID NO: 149 (ORF of the Sfr6 protein, A. thaliana), SEQ ID NO: 150 (ORF of the Shr protein, A. thaliana), SEQ ID NO : 151 (ORF of the Shy2 protein, A. thaliana), SEQ ID NO: 152 (ORF of the Ski protein, M. truncatula), SEQ ID NO: 153 (ORF of the Sps1 protein, A. thaliana), SEQ ID NO: 154 (ORF of the Spt protein, A. thaliana), SEQ ID NO: 155 (ORF of the Stn8 protein, A. thaliana), SEQ ID NO: 156 (ORF of the Tap46 protein, A. thaliana), SEQ ID NO: 157 (ORF of the Topp6 protein, A. thaliana), SEQ ID NO: 158 (ORF of the TubB6 protein, A. thaliana), SEQ ID NO: 159 (ORF of the TubB8 protein, A. thaliana), SEQ ID NO: 160 (ORF of the llbala protein, A. thaliana), SEQ ID NO: 161 (ORF of the Vim3 protein, A. thaliana), SEQ ID NO: 381 (ORF of the Sgr1 protein, A. thaliana) , SEQ ID NO: 382 (ORF of the Abi5 protein, A. thaliana), SEQ ID NO: 383 (ORF of the Hsp101 protein, A. thaliana), SEQ ID NO: 384 (ORF of the Rh10 protein, M. truncatula ) and SEQ ID NO: 385 (ORF of the Wus protein, A. thaliana).
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity. , preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1 ).
En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Par « pourcentage d’identité » entre deux séquences d’acides nucléiques (ou d’acides aminés), on entend un pourcentage de nucléotides (ou de résidus d’acides aminés) identiques entre les deux séquences à comparer, obtenu après le meilleur alignement. Ce pourcentage est purement statistique et les différences entre les deux séquences sont réparties au hasard sur toute la longueur des séquences. Le meilleur alignement (ou alignement optimal) est l’alignement pour lequel le pourcentage d’identité entre les deux séquences à comparer, comme calculé ci- après, est le plus élevé. Les comparaisons de séquences entre deux séquences d’acides nucléiques (ou d’acides aminés) sont traditionnellement réalisées en comparant ces séquences après les avoir alignées de manière optimale, ladite comparaison étant réalisée par segment ou par fenêtre de comparaison pour identifier et comparer les régions locales de similarité de séquence. L’alignement optimal des séquences pour la comparaison peut être réalisé manuellement ou au moyen d’algorithmes et de logiciels à la disposition de l’homme de l’art, par exemple, la plateforme BLAST ou le programme MatGat (Campanella, Bitincka and Smalley, 2003). By “percentage of identity” between two nucleic acid (or amino acid) sequences, we mean a percentage of nucleotides (or amino acid residues) identical between the two sequences to be compared, obtained after the best alignment. . This percentage is purely statistical and the differences between the two sequences are randomly distributed over the entire length of the sequences. The best alignment (or optimal alignment) is the alignment for which the percentage of identity between the two sequences to be compared, as calculated below, is the highest. Sequence comparisons between two nucleic acid (or amino acid) sequences are traditionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out per segment or per comparison window to identify and compare the regions local sequence similarity. The optimal alignment of sequences for comparison can be carried out manually or by means of algorithms and software available to those skilled in the art, for example, the BLAST platform or the MatGat program (Campanella, Bitincka and Smalley , 2003).
Le pourcentage d’identité entre deux séquences est déterminé en comparant ces deux séquences alignées de manière optimale par fenêtre de comparaison dans laquelle la région de la séquence à comparer peut comprendre des additions ou des délétions par rapport à la séquence de référence pour un alignement optimal entre ces deux séquences. Le pourcentage d’identité est calculé en déterminant le nombre de positions identiques pour lesquelles le nucléotide (ou l’acide aminé) est identique entre les deux séquences, en divisant ce nombre de positions identiques par le nombre total de positions dans la fenêtre de comparaison et en multipliant le résultat obtenu par 100. The percentage identity between two sequences is determined by comparing these two optimally aligned sequences by comparison window in which the region of the sequence to be compared may include additions or deletions relative to the reference sequence for optimal alignment between these two sequences. Percent identity is calculated by determining the number of identical positions for which the nucleotide (or amino acid) is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100.
Au sens de l’invention, il est entendu dans l’invention que des séquences présentant « au moins 80% d’identité » avec une séquence de référence peuvent notamment présenter au moins 80%, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % ou 100 % d’identité avec ladite séquence de référence. For the purposes of the invention, it is understood in the invention that sequences presenting “at least 80% identity” with a reference sequence may in particular present at least 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% d identity with said reference sequence.
Dans un mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. Dans un mode de réalisation, l’invention concerne également le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs : 1 to 161 and 381 to 385. In one embodiment, the invention also relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising a sequence of nucleic acids chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the method for preparing and determining an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un autre mode de réalisation, l’invention concerne le procédé de préparation et de détermination d’un altPEP tel que décrit précédemment, dans lequel la séquence dudit peptide est choisie parmi les séquences : SEQ ID NO : 224 (AtDCL1alt18), SEQ ID NO : 225 (CrDCL1alt18), SEQ ID NO : 226 (EsDCL1alt18), SEQ ID NO : 227 (AIDCL1alt19), SEQ ID NO : 228 (CsDCL1alt19), SEQ ID NO : 229 (RsDCL1alt15), SEQ ID NO : 230 (BnDCL1alt14), SEQ ID NO : 231 (BoDCL1alt15), SEQ ID NO : 232 (BRDCL1alt17), SEQ ID NO : 233 (BsEIN2alt1), SEQ ID NO : 234 (CgEIN2alt1), SEQ ID NO : 235 (CrEIN2alt1), SEQ ID NO : 236 (EsEIN2alt1), SEQ ID NO : 237 (ThEI N2alt1), SEQ ID NO : 238 (BrEIN2alt1), SEQ ID NO : 239 (BoEIN2alt1), SEQ ID NO : 240 (BnEIN2alt1), SEQ ID NO : 241 (AtEIN2alt1), SEQ ID NO : 242 (AIEI N2alt1 ), SEQ ID NO : 243 (AtAP2alt1), SEQ ID NO : 244 (AIAP2alt1), SEQ ID NO : 245 (BoAP2alt1), SEQ ID NO : 246 (CgAP2alt1), SEQ ID NO : 247 (CrAP2alt1), SEQ ID NO : 248 (BrAP2alt1), SEQ ID NO : 249 (BsAP2alt1), SEQ ID NO : 250 (EsAP2alt1), SEQ ID NO : 251 (ThAP2alt1), SEQ ID NO : 252 (altPEP_DCL1), SEQ ID NO : 253 (altPEP_CYP78A8), SEQ ID NO : 254 (altPEP_PRP39), SEQ ID NO : 255 (altPEP_PIF3), SEQ ID NO : 256 (altPEP_PIF3), SEQ ID NO : 257 (altPEP_IQD28), SEQ ID NO : 258 (altPEP_AT59), SEQ ID NO : 259 (altPEP_AT59), SEQ ID NO : 260 (altPEP_ABCG11), SEQ ID NO : 261 (altPEP_ABCG11), SEQ ID NO : 262 (altPEP_ABCG11), SEQ ID NO : 263 (altPEP_ABCG11), SEQ ID NO : 264 (altPEP_ABCG11), SEQ ID NO : 265 (altPEP_ABCG11), SEQ ID NO : 266 (altPEP_ABCG11), SEQ ID NO : 267 (altPEP_RD21), SEQ ID NO : 268 (altPEP_RD21), SEQ ID NO : 269 (altPEP_RD21), SEQ ID NO : 270 (altPEP_RD21), SEQ ID NO : 271 (altPEP_LRX2), SEQ ID NO : 272 (altPEP_LRX2), SEQ ID NO : 273 (altPEP_LRX2), SEQ ID NO : 274 (altPEP_JAC1), SEQ ID NO : 275 (altPEP_JAC1), SEQ ID NO : 276 (altPEP_JAC1), SEQ ID NO : 277 (altPEP_PSKR1), SEQ ID NO : 278 (altPEP_PSKR1), SEQ ID NO : 279 (altPEP_PSKR1), SEQ ID NO : 280 (altPEP_PSKR1), SEQ ID NO : 281 (altPEP_PSKR1), SEQ ID NO : 282 (altPEP_PSKR1), SEQ ID NO : 283 (altPEP_FVE), SEQ ID NO : 284 (altPEP_FVE), SEQ ID NO : 285 (altPEPJJBAIA), SEQ ID NO : 286 (altPEP_CIPK3), SEQ ID NO : 287 (altPEP_CIPK3), SEQ ID NO : 288 (altPEP_APG9), SEQ ID NO : 289 (altPEP_APG9), SEQ ID NO : 290 (altPEP_COI1), SEQ ID NO : 291 (altPEP_COI1), SEQ ID NO : 292 (altPEP_COI1), SEQ ID NO : 293 (altPEP_COI1), SEQ ID NO : 294 (altPEP_COI1), SEQ ID NO : 295 (altPEP_COI1), SEQ ID NO : 296 (altPEP_COI1), SEQ ID NO : 297 (altPEP_COI1), SEQ ID NO : 298 (altPEP_COI1), SEQ ID NO : 299 (altPEP_MFP2), SEQ ID NO : 300 (altPEP_MFP2), SEQ ID NO : 301 (altPEP_MFP2), SEQ ID NO : 302 (altPEP_COBL8), SEQ ID NO : 303 (altPEP_IAA31), SEQ ID NO : 304 (altPEP_CYP705A18), SEQ ID NO : 305 (altPEP_AAE16), SEQ ID NO : 306 (altPEP_AAE16), SEQ ID NO : 307 (altPEP_CYP71 B26), SEQ ID NO : 308 (altPEP_CYP71 B26), SEQ ID NO : 309 (altPEP_CYP71 B26), SEQ ID NO : 310 (altPEP_CYP71 B26), SEQ ID NO : 311 (altPEP_CYP71 B26), SEQ ID NO : 312 (altPEP_CYP71 B26), SEQ ID NO : 313 (altPEP_KP1), SEQ ID NO : 314 (altPEP_KP1), SEQ ID NO : 315 (altPEP_PEN3), SEQ ID NO : 316 (altPEP_PEN3), SEQ ID NO : 317 (altPEP_HEXO1), SEQ ID NO : 318 (altPEP_GDI2), SEQ ID NO : 319 (altPEP_GDI2), SEQ ID NO : 320 (altPEP_GDI2), SEQ ID NO : 321 (altPEP_GDI2), SEQ ID NO : 322 (altPEP_GDI2), SEQ ID NO : 323 (altPEP_SFR6), SEQ ID NO : 324 (altPEP_CRK34), SEQ ID NO : 325 (altPEP_AAE15), SEQ ID NO : 326 (altPEP_CYP97B3), SEQ ID NO : 327 (altPEP_CYP97B3), SEQ ID NO : 328 (altPEP_CYP97B3), SEQ ID NO : 329 (altPEP_CYP97B3), SEQ ID NO : 330 (altPEP_AMB2726), SEQ ID NO : 331 (altPEP_HSL1), SEQ ID NO : 332 (altPEP_HSL1), SEQ ID NO : 333 (altPEP_HSL1), SEQ ID NO : 334 (altPEP_ANAC076), SEQ ID NO : 335 (altPEP_STN8), SEQ ID NO : 336 (altPEP_EMB175), SEQ ID NO : 337 (altPEP_EMB175), SEQ ID NO : 338 (altPEP_EMB175), SEQ ID NO : 339 (altPEP_EMB175), SEQ ID NO : 340 (altPEP_EMB175), SEQ ID NO : 341 (altPEP_GCN2), SEQ ID NO : 342 (altPEP_SPS1), SEQ ID NO : 343 (altPEP_SPS1), SEQ ID NO : 344 (altPEP_SPS1), SEQ ID NO : 345 (altPEP_SPS1), SEQ ID NO : 346 (altPEP_BZO2H3), SEQ ID NO : 347 (altPEP_VIM3), SEQ ID NO : 348 (altPEP_EMB9), SEQ ID NO : 349 (altPEP_EMB9), SEQ ID NO : 350 (altPEP_RPT4A), SEQ ID NO : 351 (altPEP_TOPP6), SEQ ID NO : 352 (altPEP_DUR3), SEQ ID NO : 353 (altPEP_DUR3), SEQ ID NO : 354 (altPEP_DUR3), SEQ ID NO : 355 (altPEP_ARLB1), SEQ ID NO : 356 (altPEP_ARLB1), SEQ ID NO : 357 (altPEP_HDA18), SEQ ID NO : 358 (altPEP_HDA18), SEQ ID NO : 359 (altPEP_HDA18), SEQ ID NO : 360 (altPEP_CESA6), SEQ ID NO : 361 (altPEP_FNR1), SEQ ID NO : 362 (altPEP_FNR1), SEQ ID NO : 363 (altPEP_FNR1), SEQ ID NO : 364 (EIN2alt1), SEQ ID NO : 365 (EIN2alt2) et SEQ ID NO : 366 (EIN2alt3). Dans un deuxième aspect, l’invention ci-dessus a pour objet un altPEP tel qu’obtenu par la mise en œuvre du procédé tel que décrit précédemment. Selon ce même aspect, l’invention a également pour objet un altPEP isolé, de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment naturellement traduit d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit altPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In another embodiment, the invention relates to the method for preparing and determining an altPEP as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NO: 224 (AtDCL1alt18), SEQ ID NO: 225 (CrDCL1alt18), SEQ ID NO: 226 (EsDCL1alt18), SEQ ID NO: 227 (AIDCL1alt19), SEQ ID NO: 228 (CsDCL1alt19), SEQ ID NO: 229 (RsDCL1alt15), SEQ ID NO: 230 (BnDCL1alt14 ), SEQ ID NO: 231 (BoDCL1alt15), SEQ ID NO: 232 (BRDCL1alt17), SEQ ID NO: 233 (BsEIN2alt1), SEQ ID NO: 234 (CgEIN2alt1), SEQ ID NO: 235 (CrEIN2alt1), SEQ ID NO : 236 (EsEIN2alt1), SEQ ID NO: 237 (ThEI N2alt1), SEQ ID NO: 238 (BrEIN2alt1), SEQ ID NO: 239 (BoEIN2alt1), SEQ ID NO: 240 (BnEIN2alt1), SEQ ID NO: 241 (AtEIN2alt1 ), SEQ ID NO: 242 (AIEI N2alt1), SEQ ID NO: 243 (AtAP2alt1), SEQ ID NO: 244 (AIAP2alt1), SEQ ID NO: 245 (BoAP2alt1), SEQ ID NO: 246 (CgAP2alt1), SEQ ID NO: 247 (CrAP2alt1), SEQ ID NO: 248 (BrAP2alt1), SEQ ID NO: 249 (BsAP2alt1), SEQ ID NO: 250 (EsAP2alt1), SEQ ID NO: 251 (ThAP2alt1), SEQ ID NO: 252 (altPEP_DCL1 ), SEQ ID NO: 253 (altPEP_CYP78A8), SEQ ID NO: 254 (altPEP_PRP39), SEQ ID NO: 255 (altPEP_PIF3), SEQ ID NO: 256 (altPEP_PIF3), SEQ ID NO: 257 (altPEP_IQD28), SEQ ID NO : 258 (altPEP_AT59), SEQ ID NO: 259 (altPEP_AT59), SEQ ID NO: 260 (altPEP_ABCG11), SEQ ID NO: 261 (altPEP_ABCG11), SEQ ID NO: 262 (altPEP_ABCG11), SEQ ID NO: 263 (altPEP_ABCG11) , SEQ ID NO: 264 (altPEP_ABCG11), SEQ ID NO: 265 (altPEP_ABCG11), SEQ ID NO: 266 (altPEP_ABCG11), SEQ ID NO: 267 (altPEP_RD21), SEQ ID NO: 268 (altPEP_RD21), SEQ ID NO: 269 (altPEP_RD21), SEQ ID NO: 270 (altPEP_RD21), SEQ ID NO: 271 (altPEP_LRX2), SEQ ID NO: 272 (altPEP_LRX2), SEQ ID NO: 273 (altPEP_LRX2), SEQ ID NO: 274 (altPEP_JAC1), SEQ ID NO: 275 (altPEP_JAC1), SEQ ID NO: 276 (altPEP_JAC1), SEQ ID NO: 277 (altPEP_PSKR1), SEQ ID NO: 278 (altPEP_PSKR1), SEQ ID NO: 279 (altPEP_PSKR1), SEQ ID NO: 280 (altPEP_PSKR1), SEQ ID NO: 281 (altPEP_PSKR1), SEQ ID NO: 282 (altPEP_PSKR1), SEQ ID NO: 283 (altPEP_FVE), SEQ ID NO: 284 (altPEP_FVE), SEQ ID NO: 285 (altPEPJJBAIA), SEQ ID NO: 286 (altPEP_CIPK3), SEQ ID NO: 287 (altPEP_CIPK3), SEQ ID NO: 288 (altPEP_APG9), SEQ ID NO: 289 (altPEP_APG9), SEQ ID NO: 290 (altPEP_COI1), SEQ ID NO: 291 (altPEP_COI1), SEQ ID NO: 292 (altPEP_COI1), SEQ ID NO: 293 (altPEP_COI1), SEQ ID NO: 294 (altPEP_COI1), SEQ ID NO: 295 (altPEP_COI1), SEQ ID NO: 296 (altPEP_COI1), SEQ ID NO: 297 ( altPEP_COI1), SEQ ID NO: 298 (altPEP_COI1), SEQ ID NO: 299 (altPEP_MFP2), SEQ ID NO: 300 (altPEP_MFP2), SEQ ID NO: 301 (altPEP_MFP2), SEQ ID NO: 302 (altPEP_COBL8), SEQ ID NO: 303 (altPEP_IAA31), SEQ ID NO: 304 (altPEP_CYP705A18), SEQ ID NO: 305 (altPEP_AAE16), SEQ ID NO: 306 (altPEP_AAE16), SEQ ID NO: 307 (altPEP_CYP71 B26), SEQ ID NO: 308 ( altPEP_CYP71 B26), SEQ ID NO: 309 (altPEP_CYP71 B26), SEQ ID NO: 310 (altPEP_CYP71 B26), SEQ ID NO: 311 (altPEP_CYP71 B26), SEQ ID NO: 312 (altPEP_CYP71 B26), SEQ ID NO: 313 ( altPEP_KP1), SEQ ID NO: 314 (altPEP_KP1), SEQ ID NO: 315 (altPEP_PEN3), SEQ ID NO: 316 (altPEP_PEN3), SEQ ID NO: 317 (altPEP_HEXO1), SEQ ID NO: 318 (altPEP_GDI2), SEQ ID NO: 319 (altPEP_GDI2), SEQ ID NO: 320 (altPEP_GDI2), SEQ ID NO: 321 (altPEP_GDI2), SEQ ID NO: 322 (altPEP_GDI2), SEQ ID NO: 323 (altPEP_SFR6), SEQ ID NO: 324 (altPEP_CRK34 ), SEQ ID NO: 325 (altPEP_AAE15), SEQ ID NO: 326 (altPEP_CYP97B3), SEQ ID NO: 327 (altPEP_CYP97B3), SEQ ID NO: 328 (altPEP_CYP97B3), SEQ ID NO: 329 (altPEP_CYP97B3), SEQ ID NO : 330 (altPEP_AMB2726), SEQ ID NO: 331 (altPEP_HSL1), SEQ ID NO: 332 (altPEP_HSL1), SEQ ID NO: 333 (altPEP_HSL1), SEQ ID NO: 334 (altPEP_ANAC076), SEQ ID NO: 335 (altPEP_STN8) , SEQ ID NO: 336 (altPEP_EMB175), SEQ ID NO: 337 (altPEP_EMB175), SEQ ID NO: 338 (altPEP_EMB175), SEQ ID NO: 339 (altPEP_EMB175), SEQ ID NO: 340 (altPEP_EMB175), SEQ ID NO: 341 (altPEP_GCN2), SEQ ID NO: 342 (altPEP_SPS1), SEQ ID NO: 343 (altPEP_SPS1), SEQ ID NO: 344 (altPEP_SPS1), SEQ ID NO: 345 (altPEP_SPS1), SEQ ID NO: 346 (altPEP_BZO2H3), SEQ ID NO: 347 (altPEP_VIM3), SEQ ID NO: 348 (altPEP_EMB9), SEQ ID NO: 349 (altPEP_EMB9), SEQ ID NO: 350 (altPEP_RPT4A), SEQ ID NO: 351 (altPEP_TOPP6), SEQ ID NO: 352 (altPEP_DUR3), SEQ ID NO: 353 (altPEP_DUR3), SEQ ID NO: 354 (altPEP_DUR3), SEQ ID NO: 355 (altPEP_ARLB1), SEQ ID NO: 356 (altPEP_ARLB1), SEQ ID NO: 357 (altPEP_HDA18), SEQ ID NO: 358 (altPEP_HDA18), SEQ ID NO: 359 (altPEP_HDA18), SEQ ID NO: 360 (altPEP_CESA6), SEQ ID NO: 361 (altPEP_FNR1), SEQ ID NO: 362 (altPEP_FNR1), SEQ ID NO: 363 ( altPEP_FNR1), SEQ ID NO: 364 (EIN2alt1), SEQ ID NO: 365 (EIN2alt2) and SEQ ID NO: 366 (EIN2alt3). In a second aspect, the subject of the invention above is an altPEP as obtained by implementing the method as described above. According to this same aspect, the invention also relates to an isolated altPEP, of 4 to 70 amino acids, in particular of 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a naturally translated fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que précédemment décrit, ledit fragment ayant une taille de 3n nucléotides, n étant compris : de 4 à 41 ; de 5 à 40 ; de 7 à 20 ; ou de 8 à 15. In one embodiment, the invention relates to altPEP isolated as previously described, said fragment having a size of 3n nucleotides, n being included: from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
Autrement dit, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé comprenant s, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 ou 70 acides aminés. In other words, the invention relates to isolated altPEP as described above, said isolated altPEP comprising s, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 acids amines.
En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé comprenant de 4 à 41 acides aminés. En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé comprenant de 5 à 40 acides aminés. En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé comprenant de 7 à 20 acides aminés. En particulier, l’invention concerne également l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé comprenant de 8 à 15 acides aminés. In particular, the invention relates to isolated altPEP as described above, said isolated altPEP comprising from 4 to 41 amino acids. In particular, the invention relates to isolated altPEP as described above, said isolated altPEP comprising from 5 to 40 amino acids. In particular, the invention relates to isolated altPEP as described above, said isolated altPEP comprising from 7 to 20 amino acids. In particular, the invention also relates to isolated altPEP as described above, said isolated altPEP comprising from 8 to 15 amino acids.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel la taille dudit altPEP isolé est inférieure à celle de ladite protéine. In one embodiment, the invention relates to isolated altPEP as described above, in which the size of said isolated altPEP is less than that of said protein.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit fragment comprenant : un codon initiateur codant une méthionine initiatrice ; et un codon STOP choisie parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également l’altPEP isolé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to altPEP isolated as described above, said fragment comprising: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein. In one embodiment, the invention relates to altPEP isolated as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally occurring nucleic acid sequence. translated into said plant cell. In particular, the invention relates to altPEP isolated as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to altPEP isolated as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5' ) relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the isolated altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3 ' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ledit altPEP isolé est capable d’augmenter l’accumulation de ladite protéine dans ladite cellule végétale. In one embodiment, the invention relates to isolated altPEP as described above, wherein said isolated altPEP is capable of increasing the accumulation of said protein in said plant cell.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ledit altPEP isolé est capable diminuer l’accumulation de ladite protéine dans ladite cellule végétale. In one embodiment, the invention relates to isolated altPEP as described above, wherein said isolated altPEP is capable of reducing the accumulation of said protein in said plant cell.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ledit altPEP isolé est un peptide synthétique. In one embodiment, the invention relates to isolated altPEP as described above, wherein said isolated altPEP is a synthetic peptide.
Dans un mode de réalisation, l’invention concerne le altPEP tel que décrit précédemment, dans lequel ledit altPEP isolé est un peptide recombinant. In one embodiment, the invention relates to altPEP as described above, in which said isolated altPEP is a recombinant peptide.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé étant un peptide hydrophobe ou un peptide hydrophile. In one embodiment, the invention relates to isolated altPEP as described above, said isolated altPEP being a hydrophobic peptide or a hydrophilic peptide.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est naturellement présente dans ladite cellule végétale. In one embodiment, the invention relates to altPEP isolated as described above, in which said protein is naturally present in said plant cell.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine n’est pas présente naturellement dans ladite cellule végétale. En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un transgène introduit artificiellement dans ladite cellule végétale. En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un vecteur introduit artificiellement dans ladite cellule végétale. In one embodiment, the invention relates to altPEP isolated as described above, in which said protein is not naturally present in said plant cell. In in particular, the invention relates to isolated altPEP as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell. In particular, the invention relates to isolated altPEP as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite cellule végétale (/.e. celle dans laquelle on souhaite moduler l’accumulation d’une protéine) appartient à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the isolated altPEP as described above, in which said plant cell (/e. that in which it is desired to modulate the accumulation of a protein) belongs to a plant species chosen from : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite cellule végétale est une cellule d’une algue. In one embodiment, the invention relates to isolated altPEP as described above, in which said plant cell is a cell of an alga.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi : Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gape, Gcn2, Gdi2, G/n2, Gs/3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, \/im3, Sgr1, Abi5, Hsp101, RhIO et Wus. In one embodiment, the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl , Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726 , Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gape, Gcn2, Gdi2, G/n2, Gs/3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô , Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, \/im3, Sgr1, Abi5, Hsp101, RhIO and Wus.
En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi les gènes : Cpk3, Dell et Nsp1. En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Cpk3. En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Dell. En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par le gène Nsp1. In particular, the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene chosen from the genes: Cpk3, Dell and Nsp1. In particular, the invention relates to altPEP isolated as described above, in which said protein is encoded by the Cpk3 gene. In particular, the invention relates to altPEP isolated as described above, in which said protein is encoded by the Dell gene. Especially, the invention relates to altPEP isolated as described above, in which said protein is encoded by the Nsp1 gene.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. In one embodiment, the invention relates to isolated altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
En particulier, l’invention concerne l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). En particulier, l’invention concerne également l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne également l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne également l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. % identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1). In particular, the invention also relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention also relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention also relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. L’invention concerne notamment l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385. The invention relates in particular to altPEP isolated as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising a chosen among the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to altPEP isolated as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un autre mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel la séquence dudit peptide est choisie parmi les séquences : SEQ ID NOs : 224 à 366. In another embodiment, the invention relates to altPEP isolated as described above, in which the sequence of said peptide is chosen from the sequences: SEQ ID NOs: 224 to 366.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, dans lequel ladite protéine est impliquée dans au moins un phénotype de plante choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In one embodiment, the invention relates to altPEP isolated as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and the number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Dans l’invention, un altPEP peut être fusionné ou lié à une ou plusieurs molécules facilitant l’entrée de l’altPEP dans la cellule. Parmi ces molécules, on peut notamment citer des peptides et l’acide palmitique. Parmi ces molécules, on peut notamment citer des peptides pénétrants (Numata, K., et al. Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Sci Rep 8, 10966 (2018).) et l’acide palmitique. Par « peptide pénétrant » (ci-après CPP), on entend des peptides de petite taille pénétrant les bicouches lipidiques cellulaires ou déstabilisent les membranes cellulaires. Les CPP peuvent être classés en trois groupes : cationiques, amphipathiques et hydrophobes. En particulier : In the invention, an altPEP may be fused or linked to one or more molecules facilitating entry of the altPEP into the cell. Among these molecules, we can notably cite peptides and palmitic acid. Among these molecules, we can in particular cite penetrating peptides (Numata, K., et al. Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Sci Rep 8 , 10966 (2018).) and palmitic acid. By “penetrating peptide” (hereinafter CPP), we mean small peptides that penetrate cellular lipid bilayers or destabilize cellular membranes. CPPs can be classified into three groups: cationic, amphipathic and hydrophobic. Especially :
- les CPP cationiques contiennent de nombreux acides aminés chargés positivement, tels que la lysine (Lys) et l'arginine (Arg) ; - cationic CPPs contain many positively charged amino acids, such as lysine (Lys) and arginine (Arg);
- Les CPP amphipathiques sont généralement composés d'une séquence alternée d'acides aminés polaires et non polaires ; et - Les CPP hydrophobes se composent d'acides aminés non polaires avec des charges nettes relativement faibles. - Amphipathic CPPs are generally composed of an alternating sequence of polar and non-polar amino acids; And - Hydrophobic CPPs consist of nonpolar amino acids with relatively low net charges.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé étant fusionné à un peptide facilitant son entrée dans la cellule végétale. En particulier, l’invention concerne l’altPEP tel que décrit précédemment, ledit altPEP étant fusionné à un peptide pénétrant. In one embodiment, the invention relates to isolated altPEP as described above, said isolated altPEP being fused to a peptide facilitating its entry into the plant cell. In particular, the invention relates to altPEP as described above, said altPEP being fused to a penetrating peptide.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé étant fusionné à l’extrémité N-terminale ou à l’extrémité C-terminale avec ledit peptide facilitant son entrée dans la cellule végétale. En particulier, l’invention concerne l’altPEP tel que décrit précédemment, ledit altPEP étant fusionné à l’extrémité N-terminale ou à l’extrémité C-terminale avec ledit peptide pénétrant. In one embodiment, the invention relates to isolated altPEP as described above, said isolated altPEP being fused at the N-terminus or at the C-terminus with said peptide facilitating its entry into the plant cell. In particular, the invention relates to altPEP as described above, said altPEP being fused at the N-terminus or at the C-terminus with said penetrating peptide.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé étant fusionné avec : le peptide TAT (SEQ ID NO : 380) ; la pénétratine ; un peptide polyhistidine (notamment un peptide d’au moins 4 résidus histidine) ; ou un peptide polyarginine (notamment un peptide de 4 résidus arginine). In one embodiment, the invention relates to isolated altPEP as described above, said isolated altPEP being fused with: the TAT peptide (SEQ ID NO: 380); penetratin; a polyhistidine peptide (in particular a peptide of at least 4 histidine residues); or a polyarginine peptide (in particular a peptide of 4 arginine residues).
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé étant lié à une ou plusieurs molécules d’acide palmitique. In one embodiment, the invention relates to isolated altPEP as described above, said isolated altPEP being linked to one or more palmitic acid molecules.
Dans un mode de réalisation, l’invention concerne l’altPEP isolé tel que décrit précédemment, ledit altPEP isolé étant lié à l’extrémité N-terminale ou à l’extrémité C-terminale à une ou plusieurs molécules d’acide palmitique. In one embodiment, the invention relates to isolated altPEP as described above, said isolated altPEP being linked at the N-terminus or at the C-terminus to one or more molecules of palmitic acid.
Sur ce point, il convient de noter que la quantité de l’altPEP nécessaire pour moduler l’accumulation d’une protéine peut varier selon que l’altPEP soit ou non modifié avec l’une des molécules facilitant sa pénétration cellulaire. On this point, it should be noted that the quantity of altPEP necessary to modulate the accumulation of a protein can vary depending on whether or not altPEP is modified with one of the molecules facilitating its cellular penetration.
Dans un troisième aspect, l’invention ci-dessus a pour objet un acide nucléique codant un altPEP tel que décrit précédemment. Selon ce même aspect, l’invention a également pour objet un acide nucléique de 3n nucléotides, lequel acide nucléique correspond à un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm. In a third aspect, the invention above relates to a nucleic acid encoding an altPEP as described above. According to this same aspect, the invention also relates to a nucleic acid of 3n nucleotides, which nucleic acid corresponds to a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA.
Dans un mode de réalisation, l’invention concerne l’acide nucléique tel que décrit précédemment, ledit fragment comprenant : un codon initiateur codant une méthionine initiatrice ; et un codon STOP choisie parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the nucleic acid as described above, said fragment comprising: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne l’acide nucléique tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally occurring nucleic acid sequence. translated into said plant cell. In particular, the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in the 3' (or by two nucleotides in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5' ) relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the nucleic acid as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3 ' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
En particulier, l’invention concerne l’acide nucléique tel que décrit précédemment, où n est compris : de 4 à 70 ; de 4 à 41 ; de 5 à 40 ; de 7 à 20 ; ou de 8 à 15. In particular, the invention relates to the nucleic acid as described above, where n is comprised: from 4 to 70; from 4 to 41; from 5 to 40; from 7 to 20; or from 8 to 15.
Dans un autre aspect, l’invention ci-dessus concerne une composition comprenant un altPEP tel que décrit ci-dessus en tant que substance active. Dans un autre mode de réalisation, l’invention ci-dessus concerne une composition comprenant un altPEP en tant que substance active, ledit altPEP : In another aspect, the above invention relates to a composition comprising an altPEP as described above as an active substance. In another embodiment, the above invention relates to a composition comprising an altPEP as active substance, said altPEP:
- ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm ; et - having a size of 4 to 70 amino acids, in particular 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a naturally translated nucleic acid sequence on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA; And
- étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. - being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit fragment comprend : un codon initiateur codant une méthionine initiatrice ; et un codon STOP choisie parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the composition as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la composition telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the composition as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit altPEP est à une concentration comprise de 10'9 M à 10'3 M. Sur ce point, il convient de noter d’une part que la composition de l’invention n’existe pas à l’état naturel et ceci est d’autant plus vrai qu’une telle concentration en altPEP ne peut exister au sein d’une cellule végétale. De plus et par « concentration comprise de 10'9 M à 10'3 M », on entend que la concentration en altPEP peut être comprise de 10'9 à 10'4 M, de 10'8 à 10'4 M, de 10'9 à 10'5 M, de 10'8 à 10'5 M, comme elle peut être comprise de 5 pM à 500 pM, de 30 pM à 70 pM, ou encore être de 50 pM. In one embodiment, the invention relates to the composition as described above, in which said altPEP is at a concentration comprised from 10' 9 M to 10' 3 M. On this point, it should be noted on the one hand that the composition of the invention does not exist in the natural state and this is all the more true since such a concentration of altPEP cannot exist within of a plant cell. In addition and by "concentration comprised from 10' 9 M to 10' 3 M", it is understood that the concentration of altPEP can be comprised from 10' 9 to 10' 4 M, from 10' 8 to 10' 4 M, from 10' 9 to 10' 5 M, from 10' 8 to 10' 5 M, as it can be comprised from 5 pM to 500 pM, from 30 pM to 70 pM, or even be 50 pM.
En particulier, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit altPEP est à une concentration comprise de 10'9 à 10'4 M, de 10'8 à 10'4 M, de 10'9 à 10'5 M ou de 10'8 à 10'5 M. En particulier, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit altPEP est à une concentration comprise de 5 pM à 500 pM ou de 30 pM à 70 pM. En particulier, l’invention concerne la composition telle que décrite précédemment, dans laquelle ledit altPEP est à une concentration de 50 pM. De manière non limitative, cette concentration peut également être de 10'9 M, 10'8 M, 10'7 M, 10'6 M, IO’5 M ou IO’4 M. In particular, the invention relates to the composition as described above, in which said altPEP is at a concentration comprised from 10' 9 to 10' 4 M, from 10' 8 to 10' 4 M, from 10' 9 to 10' 5 M or from 10' 8 to 10' 5 M. In particular, the invention relates to the composition as described above, in which said altPEP is at a concentration of from 5 pM to 500 pM or from 30 pM to 70 pM. In particular, the invention relates to the composition as described above, in which said altPEP is at a concentration of 50 pM. In a non-limiting manner, this concentration can also be 10' 9 M, 10' 8 M, 10' 7 M, 10' 6 M, IO' 5 M or IO' 4 M.
Au vu de ce qui précède, on comprend que l’invention concerne également la composition comprenant un altPEP en tant que substance active, ledit altPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm ; étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine ; et étant notamment à une concentration comprise de 5 pM à 500 pM ou de 30 pM à 70 pM, ou étant notamment à une concentration de 50 pM. In view of the above, it is understood that the invention also relates to the composition comprising an altPEP as active substance, said altPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, of which the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA ; being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein; and being in particular at a concentration of 5 pM to 500 pM or from 30 pM to 70 pM, or being in particular at a concentration of 50 pM.
A noter que par « composition comprenant un altPEP », on entend que la composition de l’invention comprend au moins un altPEP. C’est-à-dire qu’un mélange d’altPEPs est envisageable, lesdits altPEPs pouvant cibler une même protéine ou plusieurs protéines selon le fragment d’acides nucléiques dont ils sont issus. A cet égard, les concentrations susmentionnées concernent soit le mélange d’altPEPs en tant que tel, soit chacun des altPEPs dudit mélange, lesdits altPEPs pouvant être à la même concentration ou pouvant être à des concentrations différentes parmi celles citées ci-dessus. Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition phytopharmaceutique, une composition herbicide ou une composition d’enrobage, en particulier ladite composition d’enrobage comprenant en outre au moins un agent de fixation. Note that by “composition comprising an altPEP”, we mean that the composition of the invention comprises at least one altPEP. That is to say that a mixture of altPEPs is possible, said altPEPs being able to target the same protein or several proteins depending on the nucleic acid fragment from which they come. In this regard, the aforementioned concentrations concern either the mixture of altPEPs as such, or each of the altPEPs of said mixture, said altPEPs being able to be at the same concentration or being able to be at different concentrations among those cited above. In one embodiment, the invention relates to the composition as described above, said composition being a phytopharmaceutical composition, a herbicidal composition or a coating composition, in particular said coating composition further comprising at least one fixing agent .
En particulier, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition phytopharmaceutique. En particulier, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition herbicide. En particulier, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition d’enrobage. De préférence, l’invention concerne la composition telle que décrite précédemment, ladite composition étant une composition d’enrobage comprenant en outre au moins un agent de fixation. In particular, the invention relates to the composition as described above, said composition being a phytopharmaceutical composition. In particular, the invention relates to the composition as described above, said composition being a herbicidal composition. In particular, the invention relates to the composition as described above, said composition being a coating composition. Preferably, the invention relates to the composition as described above, said composition being a coating composition further comprising at least one fixing agent.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un solvant. De préférence, ledit solvant est choisi parmi : l’acétone, l’acétonitrile, l’acide acétique, l’acide formique, l’adipate de diméthyle, le benzyl acétate, le bi-butyl carbonate, le dimethyl sulfoxide (DMSO), l’eau, le glutarate de diméthyle, l’hydroxyde d’ammonium, l’iso-butanol, l’iso-propanol, le lactate de diéthyle héxyle, le solvant naphta aromatique léger, le solvant naphta aromatique lourd, le succinate de diéthyle et leurs mélanges (e.g. mélange [eau ; acide acétique] ; [acétonitrile ; acide acétique], [eau, acétonitrile ; acide acétique], [eau ; DMSO], [eau ; acétonitrile] ou [eau ; hydroxyde d’ammonium]). In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one solvent. Preferably, said solvent is chosen from: acetone, acetonitrile, acetic acid, formic acid, dimethyl adipate, benzyl acetate, bi-butyl carbonate, dimethyl sulfoxide (DMSO), water, dimethyl glutarate, ammonium hydroxide, iso-butanol, iso-propanol, diethyl hexyl lactate, light aromatic solvent naphtha, heavy aromatic solvent naphtha, diethyl succinate and their mixtures (e.g. mixture [water; acetic acid]; [acetonitrile; acetic acid], [water, acetonitrile; acetic acid], [water; DMSO], [water; acetonitrile] or [water; ammonium hydroxide]) .
Les propriétés de solubilité des altPEPs sont déterminées notamment par leur composition en acides aminés. Les altPEPs hydrophiles peuvent être solubilisés et conditionnés dans des solutions aqueuses, telles que l’eau. Les altPEPs hydrophobes peuvent être solubilisés et conditionnés dans des solvants, tels que les solvants organiques. The solubility properties of altPEPs are determined in particular by their amino acid composition. Hydrophilic altPEPs can be solubilized and packaged in aqueous solutions, such as water. Hydrophobic altPEPs can be solubilized and packaged in solvents, such as organic solvents.
Pour un traitement des plantes par les altPEPs, les solvants organiques sont des solvants non toxiques pour les plantes en faibles quantités, c’est-à-dire qu’ils n’ont pas d’effet délétère sur le développement de la plante. De manière non limitative, les solvants organiques peuvent être ceux cités ci-dessus et en particulier choisis parmi l’acétonitrile et l’acide acétique. For treatment of plants with altPEPs, organic solvents are non-toxic solvents for plants in small quantities, that is to say they have no deleterious effect on the development of the plant. In a non-limiting manner, the organic solvents may be those mentioned above and in particular chosen from acetonitrile and acetic acid.
Comme indiqué ci-dessus, les altPEPs peuvent également être solubilisés et conditionnés dans des mélanges de solvants, comme par exemple, un mélange de solvant organique [acétonitrile ; acide acétique], un mélange [eau ; DMSO] dans un ratio volume:volume compris de 99:1 à 1 :99, un mélange [eau ; acétonitrile] dans un ratio volume:volume compris de 99:1 à 1 :99 ou un mélange [eau ; hydroxyde d’ammonium] dans un ratio volume:volume compris de 99:1 à 99,9:0,1. Les altPEPs peuvent également être solubilisés dans une solution comprenant 50 % d’acétonitrile, 10 % d’acide acétique et 40 % d’eau (volume/volume/volume). As noted above, altPEPs can also be solubilized and packaged in solvent mixtures, such as, for example, an organic solvent mixture [acetonitrile; acetic acid], a mixture [water; DMSO] in a volume:volume ratio of 99:1 to 1:99, a mixture [water; acetonitrile] in an inclusive volume:volume ratio of 99:1 at 1:99 or a mixture [water; ammonium hydroxide] in a volume:volume ratio of 99:1 to 99.9:0.1. AltPEPs can also be solubilized in a solution comprising 50% acetonitrile, 10% acetic acid and 40% water (volume/volume/volume).
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un diluant. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one diluent.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un adjuvant. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one adjuvant.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un agent de fixation. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one fixing agent.
Par « agent de fixation », on entend un agent chimique ou naturel lequel permet de coller la composition de l’invention à une graine de végétal de manière à enrober ladite graine de végétal. On entend également une substance rendant possible l’application et la tenue de la ou des substances actives sur le grain. Parmi les agents de fixation disponibles, on retrouve notamment la carboxymethyl cellulose (CMC) et la gomme arabique. De plus et de manière non limitative, un agent de fixation peut comprendre des solvants organiques, de l’eau, des dispersants, des émulgateurs, des tensioactifs, des mouillants et des colorants. By “fixing agent” is meant a chemical or natural agent which makes it possible to stick the composition of the invention to a plant seed so as to coat said plant seed. We also mean a substance making it possible to apply and hold the active substance(s) on the grain. Among the fixing agents available, we find in particular carboxymethyl cellulose (CMC) and gum arabic. Additionally and without limitation, a fixing agent may include organic solvents, water, dispersants, emulgators, surfactants, wetting agents and dyes.
Dans un mode de réalisation, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un nutriment végétal. En particulier, l’invention concerne la composition telle que décrite précédemment, ladite composition comprenant en outre au moins un agent de fixation et au moins un nutriment végétal. In one embodiment, the invention relates to the composition as described above, said composition further comprising at least one plant nutrient. In particular, the invention relates to the composition as described above, said composition further comprising at least one fixing agent and at least one plant nutrient.
Par « nutriment végétal », on entend un élément assimilé par la plante pour permettre son développement. De manière non limitative, un nutriment végétal peut être choisi parmi : l’azote, le phosphore, le potassium, le calcium, le magnésium, le soufre, le manganèse, le fer, le cuivre, le bore, le zinc, le molybdène et leurs mélanges. By “plant nutrient”, we mean an element assimilated by the plant to enable its development. In a non-limiting manner, a plant nutrient can be chosen from: nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, manganese, iron, copper, boron, zinc, molybdenum and their mixtures.
Au vu de ce qui précède, on comprend qu’un autre aspect de l’invention a pour objet une semence enrobée comprenant une graine de plante, ladite graine de plante étant enrobée par une composition d’enrobage telle que décrite précédemment. L’enrobage peut être réalisé selon les procédés classiquement utilisés dans l’industrie agroalimentaire et peut être obtenu en utilisant un matériau apte à se désagréger dans un solvant ou dans la terre, tel qu’un liant ou de l’argile. In view of the above, it is understood that another aspect of the invention relates to a coated seed comprising a plant seed, said plant seed being coated with a coating composition as described above. The coating can be carried out according to the methods conventionally used in the food industry and can be obtained by using a material capable of disintegrating in a solvent or in the earth, such as a binder or clay.
Selon l’invention, l’enrobage peut être utilisé pour conférer des propriétés particulières à une semence en combinaison avec un altPEP, telles qu’une croissance améliorée ou une résistance à certains stress biotiques ou abiotiques. According to the invention, the coating can be used to confer particular properties to a seed in combination with an altPEP, such as improved growth or resistance to certain biotic or abiotic stresses.
Dans un mode de réalisation, l’invention concerne la semence enrobée telle que décrite précédemment, dans lequel ladite graine de plante à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the coated seed as described above, in which said plant seed has a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne la semence enrobée telle que décrite précédemment, ladite semence étant traitée par trempage dans une composition contenant un altPEP. Lors d’un trempage, la semence est alors plongée totalement ou partiellement dans une composition contenant un altPEP. In one embodiment, the invention relates to coated seed as described above, said seed being treated by soaking in a composition containing an altPEP. During soaking, the seed is then totally or partially immersed in a composition containing an altPEP.
Dans un autre aspect, l’invention concerne une utilisation d’un altPEP en tant qu’agent phytosanitaire pour moduler l’accumulation d’une protéine dans une cellule végétale, ledit altPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit altPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit fragment comprend : un codon initiateur codant une méthionine initiatrice ; et un codon STOP choisie parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In another aspect, the invention relates to a use of an altPEP as a phytosanitary agent to modulate the accumulation of a protein in a plant cell, said altPEP having a size of 4 to 70 amino acids, in particular from 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein. In one embodiment, the invention relates to the use of an altPEP as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the use of an altPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the sequence of nucleic acids naturally translated in said plant cell. In particular, the invention relates to the use of an altPEP as described previously, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the use of an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in 3' (or by one nucleotide in 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the use of an altPEP as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment pour augmenter l’accumulation de ladite protéine dans la cellule végétale. La présence de l’altPEP fait que la quantité de ladite protéine dans la cellule végétale traitée est supérieure à celle dans une cellule végétale non traitée. In one embodiment, the invention relates to the use of an altPEP as described above to increase the accumulation of said protein in the plant cell. The presence of altPEP causes the quantity of said protein in the treated plant cell to be greater than that in an untreated plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment pour diminuer (inhiber) l’accumulation de ladite protéine dans la cellule végétale. La présence du altPEP fait que la quantité de ladite protéine dans la cellule végétale traitée est inférieure à celle dans une cellule végétale non traitée. In one embodiment, the invention relates to the use of an altPEP as described above to reduce (inhibit) the accumulation of said protein in the plant cell. The presence of altPEP causes the quantity of said protein in the treated plant cell to be lower than that in an untreated plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP est produit hors de ladite cellule végétale avant d’être introduit dans ladite cellule végétale. Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP est un peptide synthétique. In one embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP is produced outside of said plant cell before being introduced into said plant cell. In one embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP is a synthetic peptide.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP est un peptide isolé. In one embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP is an isolated peptide.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP est un peptide recombinant. In one embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP is a recombinant peptide.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP est un peptide hydrophobe ou un peptide hydrophile. In one embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP is a hydrophobic peptide or a hydrophilic peptide.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP est introduit dans ladite cellule végétale sous la forme d’un acide nucléique codant ledit altPEP. En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP est introduit dans ladite cellule végétale sous la forme d’un acide nucléique codant ledit altPEP et comprenant les moyens de l’exprimer. In one embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP is introduced into said plant cell in the form of a nucleic acid encoding said altPEP. In particular, the invention relates to the use of an altPEP as described above, in which said altPEP is introduced into said plant cell in the form of a nucleic acid encoding said altPEP and comprising the means of expressing it.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est naturellement présente dans ladite cellule végétale. In one embodiment, the invention relates to the use of an altPEP as described above, in which said protein is naturally present in said plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine n’est pas présente naturellement dans ladite cellule végétale. En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un transgène introduit artificiellement dans ladite cellule végétale. En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un vecteur introduit artificiellement dans ladite cellule végétale. In one embodiment, the invention relates to the use of an altPEP as described above, in which said protein is not naturally present in said plant cell. In particular, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell. In particular, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle l’accumulation de ladite protéine est déterminée via la mise en œuvre d’une technique choisie parmi : le Western blot, la mesure de l’activité enzymatique, la spectrométrie de masse et la fusion traductionnelle. En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle l’accumulation de ladite protéine est déterminée via la mise en œuvre d’un Western blot. Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP a une taille comprise de 4 à 41 acides aminés, de 5 à 40 acides aminés, de 7 à 20 acides aminés ou plus particulièrement une taille comprise de 8 à 15 acides aminés. En particulier, ledit altPEP a une taille de 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 ou 70 acides aminés. In one embodiment, the invention relates to the use of an altPEP as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blotting, measurement of enzymatic activity, mass spectrometry and translational fusion. In particular, the invention relates to the use of an altPEP as described above, in which the accumulation of said protein is determined via the implementation of a Western blot. In one embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP has a size comprised from 4 to 41 amino acids, from 5 to 40 amino acids, from 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids. In particular, said altPEP has a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite cellule végétale appartient à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the use of an altPEP as described above, in which said plant cell belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite cellule végétale est une cellule d’une algue. In particular, the invention relates to the use of an altPEP as described above, in which said plant cell is a cell of an alga.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène choisi parmi : Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 et Wus. In one embodiment, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Arlbl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, Gln2, Gsl3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppo, TubB6, TubB8, Ubala, Vim3, Sgr1, Abi5, Hsp101, Rh10 and Wus.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. In one embodiment, the invention relates to the use of an altPEP as described above, in which said protein is encoded by an ORF comprising a sequence of nucleic acids having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the use of an altPEP as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90%. identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. Dans un mode de réalisation, l’invention concerne également l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385. In one embodiment, the invention also relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the use of an altPEP as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un autre mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ledit altPEP est choisi parmi les séquences : SEQ ID NOs : 224 à 366. In another embodiment, the invention relates to the use of an altPEP as described above, in which said altPEP is chosen from the sequences: SEQ ID NOs: 224 to 366.
Dans un autre mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, dans laquelle ladite protéine est impliquée dans au moins un phénotype de plante choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In another embodiment, the invention relates to the use of an altPEP as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume , the mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Dans un autre mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, pour moduler l’accumulation d’une protéine recombinante dont la séquence d’acides nucléiques qui la code correspond à la fusion des séquences d’acides nucléiques de deux gènes distincts. In another embodiment, the invention relates to the use of an altPEP as described above, to modulate the accumulation of a recombinant protein whose nucleic acid sequence which encodes it corresponds to the fusion of sequences d nucleic acids of two distinct genes.
En particulier, la séquence codante d’au moins un des deux gènes est celle d’un gène rapporteur, par exemple un gène codant une protéine fluorescente (telle que la GFP) ou une protéine permettant la résistance de la plante à un composé. In particular, the coding sequence of at least one of the two genes is that of a reporter gene, for example a gene encoding a fluorescent protein (such as GFP) or a protein allowing resistance of the plant to a compound.
Dans un mode de réalisation, l’invention concerne l’utilisation d’un altPEP telle que décrite précédemment, pour moduler l’accumulation d’une protéine recombinante dont la séquence d’acides nucléiques qui la code correspond à la fusion : d’une séquence d’acides nucléiques réputée non codante d’un premier gène ; et d’une séquence d’acides nucléiques codante d’un second gène, la séquence dudit altPEP correspondant à la traduction via le code génétique d’un fragment de la séquence d’acides nucléiques réputée non codante du premier gène. In one embodiment, the invention relates to the use of an altPEP as described above, to modulate the accumulation of a recombinant protein whose nucleic acid sequence which encodes it corresponds to the fusion: of a nucleic acid sequence deemed non-coding of a first gene; And of a coding nucleic acid sequence of a second gene, the sequence of said altPEP corresponding to the translation via the genetic code of a fragment of the nucleic acid sequence deemed non-coding of the first gene.
Dans un autre aspect, l’invention ci-dessus concerne un procédé de modulation de l’accumulation d’une protéine dans une cellule végétale comprenant une étape d’introduction : d’un altPEP ; ou d’un acide nucléique codant ledit altPEP et les moyens de l’exprimer, dans ladite cellule végétale, l’introduction dudit altPEP entraînant une modulation de la quantité de ladite protéine dans ladite cellule végétale, ledit altPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit altPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In another aspect, the above invention relates to a method of modulating the accumulation of a protein in a plant cell comprising a step of introducing: an altPEP; or a nucleic acid encoding said altPEP and the means of expressing it, in said plant cell, the introduction of said altPEP leading to a modulation of the quantity of said protein in said plant cell, said altPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit fragment comprenant : un codon initiateur codant une méthionine initiatrice ; et un codon STOP choisie parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the method as described above, in which said fragment comprising: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne le procédé tel que décrit précédemment, dans lequel la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide in 3' (or two nucleotides in 5') relative to to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') by relation to the frame open for reading the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the method as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides at 5') or two nucleotides at 3' ( or a nucleotide 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, ledit procédé permettant : de favoriser le développement d’une plante ; ou de ralentir ou d’empêcher le développement d’une plante. In one embodiment, the invention relates to the method as described above, said method making it possible to: promote the development of a plant; or slow down or prevent the development of a plant.
En particulier, l’invention concerne le procédé tel que décrit précédemment, ledit procédé permettant de favoriser le développement d’une plante. En particulier, l’invention concerne le procédé tel que décrit précédemment, ledit procédé permettant de ralentir ou d’empêcher le développement d’une plante. In particular, the invention relates to the process as described above, said process making it possible to promote the development of a plant. In particular, the invention relates to the process as described above, said process making it possible to slow down or prevent the development of a plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment pour augmenter l’accumulation de ladite protéine dans la cellule végétale. La présence du altPEP fait que la quantité de ladite protéine dans la cellule végétale traitée est supérieure à celle dans une cellule végétale non traitée. In one embodiment, the invention relates to the method as described above for increasing the accumulation of said protein in the plant cell. The presence of altPEP causes the quantity of said protein in the treated plant cell to be greater than that in an untreated plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment pour diminuer (inhiber) l’accumulation de ladite protéine dans la cellule végétale. La présence du altPEP fait que la quantité de ladite protéine dans la cellule végétale traitée est inférieure à celle dans une cellule végétale non traitée. In one embodiment, the invention relates to the method as described above for reducing (inhibiting) the accumulation of said protein in the plant cell. The presence of altPEP causes the quantity of said protein in the treated plant cell to be lower than that in an untreated plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est produit hors de ladite cellule végétale avant d’être introduit dans ladite cellule végétale. In one embodiment, the invention relates to the method as described above, in which said altPEP is produced outside of said plant cell before being introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est un peptide synthétique. In one embodiment, the invention relates to the method as described above, in which said altPEP is a synthetic peptide.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est un peptide isolé. In one embodiment, the invention relates to the method as described above, in which said altPEP is an isolated peptide.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est un peptide recombinant. Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est un peptide hydrophobe ou un peptide hydrophile. In one embodiment, the invention relates to the method as described above, in which said altPEP is a recombinant peptide. In one embodiment, the invention relates to the method as described above, in which said altPEP is a hydrophobic peptide or a hydrophilic peptide.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est introduit dans ladite cellule végétale sous la forme d’un acide nucléique codant ledit altPEP. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est introduit dans ladite cellule végétale sous la forme d’un acide nucléique codant ledit altPEP et comprenant les moyens de l’exprimer. In one embodiment, the invention relates to the method as described above, in which said altPEP is introduced into said plant cell in the form of a nucleic acid encoding said altPEP. In particular, the invention relates to the method as described above, in which said altPEP is introduced into said plant cell in the form of a nucleic acid encoding said altPEP and comprising the means of expressing it.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est naturellement présente dans ladite cellule végétale. In one embodiment, the invention relates to the method as described above, in which said protein is naturally present in said plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine n’est pas présente naturellement dans ladite cellule végétale. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un transgène introduit artificiellement dans ladite cellule végétale. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un vecteur introduit artificiellement dans ladite cellule végétale. In one embodiment, the invention relates to the method as described above, in which said protein is not naturally present in said plant cell. In particular, the invention relates to the method as described above, in which said protein is encoded by a transgene artificially introduced into said plant cell. In particular, the invention relates to the method as described above, in which said protein is encoded by a vector artificially introduced into said plant cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’accumulation de ladite protéine est déterminée via la mise en œuvre d’une technique choisie parmi : le Western blot, la mesure de l’activité enzymatique, la spectrométrie de masse et la fusion traductionnelle. En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’accumulation de ladite protéine est déterminée via la mise en œuvre d’un Western blot. In one embodiment, the invention relates to the method as described above, in which the accumulation of said protein is determined via the implementation of a technique chosen from: Western blotting, measurement of enzymatic activity , mass spectrometry and translational fusion. In particular, the invention relates to the method as described above, in which the accumulation of said protein is determined via the implementation of a Western blot.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP a une taille comprise de 4 à 41 acides aminés, de 5 à 40 acides aminés, de 7 à 20 acides aminés ou plus particulièrement une taille comprise de 8 à 15 acides aminés. En particulier, ledit altPEP a une taille de 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 ou 70 acides aminés. In one embodiment, the invention relates to the method as described above, in which said altPEP has a size comprised from 4 to 41 amino acids, from 5 to 40 amino acids, from 7 to 20 amino acids or more particularly a size comprised of 8 to 15 amino acids. In particular, said altPEP has a size of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite cellule végétale ou ladite plante appartient à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the method as described above, in which said plant cell or said plant belongs to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata , Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii , Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite cellule végétale est une cellule d’une algue. In particular, the invention relates to the method as described above, in which said plant cell is an algae cell.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène choisi parmi : Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Aribl, Arr1, Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9, Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, G/n2, Gs/3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2, Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, \/im3, Sgr1, Abi5, Hsp101, RhIO et Wus. In one embodiment, the invention relates to the method as described above, in which said protein is encoded by a gene chosen from: Aae15, Aae16, abcg11, Abdcg34, Acc1, Agb1, Als, Anac076, Apg9, Aribl, Arr1 , Arr5, Arr6, At59, Bak1, Bccpl, Bccp2, Bri1, Bzo2h3, Cesaô, Cipk3, Cks1, Cobl8, Coi1, Cpk3, Crk34, Cyp705a18, Cyp71b26, Cyp78a8, Cyp97b3, Dell, Dur3, Ein2, Emb175, Emb2726, Emb9 , Epsps, Fnr1, Eve, Ga2ox7, Gapc, Gcn2, Gdi2, G/n2, Gs/3, Hag5, Hda18, Hexol, Hppd, Hsl1, Iaa31, Iqd28, Jac1, Jar1, Kp1, Lrx2, Mapkkk3, Mapkkkô, Mfp2 , Mrb1, Nsp1, Pds, Pen3, Phyb, Pif 3, Pizza, Ppoxl, Ppox2, Prp39, PsbA, Pskrl, Rd21, Ringl, Rosi, Rpt4a, Sfr6, Shr, Shy2, Ski, Sps1, Spt, Stn8, Tap46, Toppô, TubB6, TubB8, Ubala, \/im3, Sgr1, Abi5, Hsp101, RhIO and Wus.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. In one embodiment, the invention relates to the method as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un ORF comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the method as described above, in which said protein is encoded by an ORF comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity. identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques présentant au moins 80 % d’identité, de préférence au moins 90 % d’identité, avec une séquence choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence having at least 80% identity, preferably at least 90% identity, with a sequence chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 1 à 161 et 381 à 385. Dans un mode de réalisation, l’invention concerne également le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une séquence d’acides nucléiques choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3), SEQ ID NOs : 61 à 79 (Dell) et SEQ ID NOs : 109 à 134 (Nsp1). In one embodiment, the invention relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 1 to 161 and 381 to 385. In one embodiment, the invention also relates to the method as described above, in which said protein is encoded by a gene comprising a nucleic acid sequence chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3), SEQ ID NOs: 61 to 79 (Dell) and SEQ ID NOs: 109 to 134 (Nsp1).
En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 26 à 55 (Cpk3). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 61 à 79 (Dell). En particulier, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est codée par un gène comprenant une choisie parmi les séquences : SEQ ID NOs : 109 à 134 (Nsp1). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 26 to 55 (Cpk3). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 61 to 79 (Dell). In particular, the invention relates to the method as described above, in which said protein is encoded by a gene comprising one chosen from the sequences: SEQ ID NOs: 109 to 134 (Nsp1).
Dans un autre mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est choisi parmi les séquences : SEQ ID NOs : 224 à 366. In another embodiment, the invention relates to the method as described above, in which said altPEP is chosen from the sequences: SEQ ID NOs: 224 to 366.
Dans un autre mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ladite protéine est impliquée dans au moins un phénotype de plante choisi parmi : la taille, la forme, la surface, le volume, la masse et le nombre de feuilles ; la taille, la forme, la surface, le volume, la masse et le nombre de fleurs ; la taille de la tige (ou de la hampe florale) ; la biomasse racinaire ; le nombre, la longueur et le niveau de ramification des racines ; la précocité de la germination ; la précocité du bourgeonnement ; la précocité de l’induction florale (ou transition florale) ; la vigueur germinative et la durée de phase juvénile ; la durée de la floraison ; la résistance à un stress biotique ; la résistance à un stress abiotique ; et le nombre de cellules. In another embodiment, the invention relates to the method as described above, in which said protein is involved in at least one plant phenotype chosen from: size, shape, surface area, volume, mass and number of leaves; the size, shape, surface area, volume, mass and number of flowers; the size of the stem (or flower stalk); root biomass; the number, length and level of branching of roots; the precocity of germination; the precocity of budding; the precocity of floral induction (or floral transition); germinal vigor and duration of juvenile phase; the duration of flowering; resistance to biotic stress; resistance to abiotic stress; and the number of cells.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’introduction dudit altPEP entraîne une précocité de la montaison chez ladite plante. In one embodiment, the invention relates to the method as described above, in which the introduction of said altPEP results in early bolting in said plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’introduction dudit altPEP entraîne une précocité de la floraison chez ladite plante. In one embodiment, the invention relates to the method as described above, in which the introduction of said altPEP results in earlier flowering in said plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’introduction dudit altPEP entraîne une augmentation de la taille de la tige chez ladite plante. In one embodiment, the invention relates to the method as described above, in which the introduction of said altPEP causes an increase in the size of the stem in said plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel l’introduction dudit altPEP entraîne une précocité de croissance de la tige chez ladite plante. In one embodiment, the invention relates to the method as described above, in which the introduction of said altPEP results in earlier growth of the stem in said plant.
Les Inventeurs ont en effet constaté de manière inattendue qu’il est possible d’appliquer directement un altPEP sur la plante, e.g. via l’emploi de la composition de l’invention (cf. supra) comprenant un altPEP, pour moduler l’accumulation d’une protéine cible dans la plante, ce qui indique que le altPEP est capté par la plante. The inventors have in fact unexpectedly observed that it is possible to directly apply an altPEP to the plant, e.g. via the use of the composition of the invention (see above) comprising an altPEP, to modulate the accumulation of a target protein in the plant, indicating that altPEP is taken up by the plant.
Par conséquent, dans un mode de réalisation, l’invention concerne le procédé tel que décrit précédemment, dans lequel ledit altPEP est introduit dans ladite plante : par arrosage, par pulvérisation ou par l’ajout d’un engrais, d’un terreau, d’un substrat de culture ou d’un support en contact avec la plante, ledit altPEP étant notamment administré à la plante sous la forme d’une composition comprenant de IO’9 M à IO’4 M dudit altPEP ; par arrosage, par trempage, par pulvérisation ou par l’ajout d’un engrais, d’un terreau, d’un substrat de culture ou d’un support en contact avec la plante, ledit altPEP étant notamment administré à une graine ou une semence sous la forme d’une composition comprenant de 10'9 M à 10'4 M dudit altPEP ; ou par le biais d’un acide nucléique codant ledit altPEP et comprenant les moyen d’exprimer ledit altPEP, ledit acide nucléique étant introduit artificiellement dans la plante. Consequently, in one embodiment, the invention relates to the method as described above, in which said altPEP is introduced into said plant: by watering, by spraying or by adding a fertilizer, a potting soil, a culture substrate or a support in contact with the plant, said altPEP being in particular administered to the plant in the form of a composition comprising from IO' 9 M to IO' 4 M of said altPEP; by watering, by soaking, by spraying or by adding a fertilizer, a potting soil, a growing substrate or a support in contact with the plant, said altPEP being in particular administered to a seed or a seed in the form of a composition comprising from 10'9 M to 10'4 M of said altPEP; Or by means of a nucleic acid encoding said altPEP and comprising the means of expressing said altPEP, said nucleic acid being artificially introduced into the plant.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel ledit altPEP est introduit artificiellement par voie externe dans la plante, de préférence par arrosage, par pulvérisation ou par l’ajout d’un engrais, d’un terreau, d’un substrat de culture ou d’un support inerte. In one embodiment, the invention relates to the method as defined above, in which said altPEP is artificially introduced externally into the plant, preferably by watering, by spraying or by the addition of a fertilizer, potting soil, a growing substrate or an inert support.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel ledit altPEP est introduit par arrosage. In one embodiment, the invention relates to the method as defined above, in which said altPEP is introduced by watering.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel ledit altPEP est introduit par pulvérisation. In one embodiment, the invention relates to the method as defined above, in which said altPEP is introduced by spraying.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel ledit altPEP est introduit par l’ajout d’un engrais. In one embodiment, the invention relates to the process as defined above, in which said altPEP is introduced by the addition of a fertilizer.
Dans un mode de réalisation, l’invention concerne le procédé tel que défini précédemment, dans lequel la plante est traitée avec une composition comprenant de 10'9 M à 10'4 M dudit altPEP, ou comprenant notamment 10'9 M, 10'8 M, 10'7 M, 10'6 M, 10'5 M ou 10'4 M dudit altPEP. De préférence, les compositions ont une concentration de 10'8 M à 10'5 M pour une application par arrosage ou par pulvérisation sur la plante. In one embodiment, the invention relates to the process as defined above, in which the plant is treated with a composition comprising from 10' 9 M to 10' 4 M of said altPEP, or notably comprising 10' 9 M, 10' 8 M, 10' 7 M, 10' 6 M, 10' 5 M or 10' 4 M of said altPEP. Preferably, the compositions have a concentration of 10'8 M to 10'5 M for application by watering or spraying on the plant.
De manière complémentaire, des compositions plus ou moins concentrées peuvent être envisagées pour traiter la plante avec le altPEP. Par exemple, et de manière non limitative, des compositions plus concentrées comprenant de 10'1 M à 10'3 M, ou comprenant notamment 10'2 M de altPEP, peuvent être utilisées dans le cas où le altPEP introduit artificiellement par voie externe est administré à la plante par épandage. In addition, more or less concentrated compositions can be considered to treat the plant with altPEP. For example, and in a non-limiting manner, more concentrated compositions comprising from 10' 1 M to 10' 3 M, or notably comprising 10' 2 M of altPEP, can be used in the case where the altPEP artificially introduced externally is administered to the plant by spreading.
Dans un autre aspect, l’invention ci-dessus concerne une plante modifiée contenant un altPEP, laquelle « plante modifiée » correspond à une plante dans laquelle a été introduit artificiellement un altPEP, notamment par arrosage, par pulvérisation ou via un engrais. In another aspect, the above invention relates to a modified plant containing an altPEP, which “modified plant” corresponds to a plant into which an altPEP has been artificially introduced, in particular by watering, by spraying or via fertilizer.
Dans un mode de réalisation, l’invention concerne la plante modifiée comprenant un altPEP introduit par voie exogène, ledit altPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit altPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In one embodiment, the invention relates to the modified plant comprising an exogenously introduced altPEP, said altPEP having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle ledit fragment comprend : un codon initiateur codant une méthionine initiatrice ; et un codon STOP choisie parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the modified plant as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la plante modifiée telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally translated nucleic acid sequence. in said plant cell. In particular, the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the modified plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in the 5') or two nucleotides in the 3' (or a nucleotide at 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne la plante modifiée telle que décrite précédemment, ladite plante appartenant à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the modified plant as described above, said plant belonging to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato) , Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un autre aspect, l’invention ci-dessus concerne une plante transgénique comprenant un acide nucléique codant un altPEP et les moyens de l’exprimer, ledit altPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit altPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. In another aspect, the above invention relates to a transgenic plant comprising a nucleic acid encoding an altPEP and the means for expressing it, said altPEP having a size of from 4 to 70 amino acids, in particular from 4 to 41 acids. amino, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of an encoded protein by said mRNA, said altPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle ledit fragment comprend : un codon initiateur codant une méthionine initiatrice ; et un codon STOP choisie parmi les codons : UAG, UGA et UAA, et ledit fragment étant choisi dans un cadre ouvert de lecture décalé d’un ou deux nucléotides par rapport à celui codant ladite protéine. In one embodiment, the invention relates to the transgenic plant as described above, in which said fragment comprises: an initiator codon encoding an initiator methionine; and a STOP codon chosen from the codons: UAG, UGA and UAA, and said fragment being chosen in an open reading frame shifted by one or two nucleotides relative to that encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture différent du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un nucléotide en 3’ (ou de deux nucléotides en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. En particulier, l’invention concerne également la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé de deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. Autrement dit, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle la traduction du fragment de l’ARNm est opérée dans un cadre de lecture décalé d’un (ou deux nucléotides en 5’) ou deux nucléotides en 3’ (ou d’un nucléotide en 5’) par rapport au codon d’initiation du cadre ouvert de lecture de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale. In one embodiment, the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame different from the open reading frame of the naturally translated nucleic acid sequence. in said plant cell. In particular, the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one nucleotide at 3' (or two nucleotides at 5') by relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In particular, the invention also relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by two nucleotides in the 3' (or by one nucleotide in the 5') relative to the open reading frame of the nucleic acid sequence naturally translated in said plant cell. In other words, the invention relates to the transgenic plant as described above, in which the translation of the mRNA fragment is carried out in a reading frame shifted by one (or two nucleotides in 5') or two nucleotides in 3' (or one nucleotide in 5') relative to the initiation codon of the open reading frame of the nucleic acid sequence naturally translated in said plant cell.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que définie précédemment, dans laquelle la séquence codant ledit altPEP est plus courte que la séquence de l’ARNm codant ladite protéine. In one embodiment, the invention relates to the transgenic plant as defined above, in which the sequence encoding said altPEP is shorter than the sequence of the mRNA encoding said protein.
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, ladite plante appartenant à une espèce végétale choisie parmi : Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (colza), Brassica oleracea, Brassica rapa (colza), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soja), Gossypium raimondii, Gossypium spp. (coton), Hordeum vulgare (orge), Lollium spp., Lotus japonicus (lotier), Medicago sativa (luzerne), Medicago truncatula (luzerne), Nicotiana benthamiana (tabac), Oryza sativa (riz), Pisum sativum (pois), Raphanus sativus, Solanum lycopersicum (tomate), Solanum melongena (aubergine), Solanum tuberosum (pomme de terre), Thellungiella halophila, Theobroma cacao, Triticum spp. (blé), Vitis vinifera (vigne) et Zea mays (maïs). In one embodiment, the invention relates to the transgenic plant as described above, said plant belonging to a plant species chosen from: Alopecurus myosuroides, Amaranthus hypochondriacus, Amaranthus palmeri, Amaranthus tuberculatus, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis lyrata, Arabidopsis thaliana, Barbarea vulgaris, Boechera stricta, Brachypodium distachyon, Brassica napus (rapeseed), Brassica oleracea, Brassica rapa (rapeseed), Camelina sativa, Capsella grandiflora, Capsella rubella, Carica papaya, Eutrema salsugineum, Glycine max (soybean), Gossypium raimondii, Gossypium spp. (cotton), Hordeum vulgare (barley), Lollium spp., Lotus japonicus (trefoil), Medicago sativa (alfalfa), Medicago truncatula (alfalfa), Nicotiana benthamiana (tobacco), Oryza sativa (rice), Pisum sativum (pea), Raphanus sativus, Solanum lycopersicum (tomato), Solanum melongena (eggplant), Solanum tuberosum (potato), Thellungiella halophila, Theobroma cacao, Triticum spp. (wheat), Vitis vinifera (vine) and Zea mays (corn).
Dans un mode de réalisation, l’invention concerne la plante transgénique telle que décrite précédemment, dans laquelle l’expression dudit altPEP est placée sous le contrôle d’un promoteur fort, de préférence un promoteur fort constitutif tel que le promoteur 35S. In one embodiment, the invention relates to the transgenic plant as described above, in which the expression of said altPEP is placed under the control of a strong promoter, preferably a strong constitutive promoter such as the 35S promoter.
A tout égard, il convient de noter que les différents aspects de l’invention No. 2, tout comme les différents modes de réalisation de celle-ci sont interdépendants. Ces derniers peuvent donc être combinés entre eux pour obtenir des aspects et/ou des modes de réalisation préférés de l’invention No. 2 non explicitement décrits. Ceci est également valable pour l’ensemble des définitions fourni dans la présente description, lequel s’applique à tous les aspects de l’invention No. 2 et ses modes de réalisation. En outre, les inventions No. 1 et No. 2 sont illustrées, sans toutefois s’y limiter, par les Figures et Exemples suivants. In all respects, it should be noted that the different aspects of invention No. 2, just like the different embodiments thereof, are interdependent. The latter can therefore be combined with each other to obtain preferred aspects and/or embodiments of invention No. 2 not explicitly described. This is also valid for all definitions provided in this description, which applies to all aspects of Invention No. 2 and its embodiments. Furthermore, inventions No. 1 and No. 2 are illustrated, but not limited to, the following Figures and Examples.
LISTE DES FIGURES LIST OF FIGURES
Figure 1 Figure 1
La figure 1 est une représentation schématique illustrant les premières étapes du procédé de préparation et de détermination d’un cPEP. En particulier sont illustrés les étapes permettant de déterminer au sein d’un ARNm d’une protéine l’une des séquences d’acides nucléiques à partir de laquelle le peptide à tester (/.e. le cPEP potentiel) est identifié. Figure 1 is a schematic representation illustrating the first steps of the process for preparing and determining a cPEP. In particular, the steps making it possible to determine within an mRNA of a protein one of the nucleic acid sequences from which the peptide to be tested (/e. the potential cPEP) is identified are illustrated.
Les séquences : SEQ ID NOs : 375 à 379 sont uniquement fournies à titre d’exemple. The sequences: SEQ ID NOs: 375 to 379 are provided as an example only.
Figure 2 Figure 2
Identification et caractérisation des altPEPs Identification and characterization of altPEPs
(a) Représentation schématique d'un gène codant portant des altORFs produisant des altPEPs. (b) Distribution du classement des ORFs des altPEPs détectés par MS. (c) Fréquence des altPEPs détectés par MS par plages de longueur, (d) Longueur de la racine principale des plantules d'Æ thaliana traitées avec 100 pM de peptide pendant 4 jours, (e, f) Quantification de l'expression de DCL1 par RT-qPCR (e) et par Western blot en utilisant des anticorps anti-DCL1 (f) dans des feuilles de A. thaliana traitées pendant 5 jours avec 100 pM de peptide, (g, h) Quantification de COI1 , AP2, CPK3 (g) et EIN2 (h) par Western blot en utilisant des anticorps spécifiques dans des feuilles de A. thaliana traitées pendant 5 jours avec 100 pM de peptide. (a) Schematic representation of a gene encoding altORFs producing altPEPs. (b) ORF ranking distribution of altPEPs detected by MS. (c) Frequency of altPEPs detected by MS by length ranges, (d) Length of the main root of Æ thaliana seedlings treated with 100 pM peptide for 4 days, (e, f) Quantification of DCL1 expression by RT-qPCR (e) and by Western blot using anti-DCL1 antibodies (f) in A. thaliana leaves treated for 5 days with 100 pM peptide, (g, h) Quantification of COI1, AP2, CPK3 (g) and EIN2 (h) by Western blotting using specific antibodies in A. thaliana leaves treated for 5 days with 100 pM peptide.
Les résultats sont représentatifs de quatre expériences indépendantes. Les chiffres indiquent la moyenne et le SEM. Les témoins sont le peptide non pertinent et l'eau. Les barres d'erreur représentent les SEM, les astérisques indiquent une différence significative entre la condition de test et le contrôle selon le test t de Student (d) ou le test de Wilcoxon (e-h) (n = 100 (d), n = 4 (e-h) ; p < 0,05). Results are representative of four independent experiments. Numbers indicate mean and SEM. The controls are the irrelevant peptide and water. Error bars represent SEMs, asterisks indicate significant difference between test condition and control according to Student's t test (d) or Wilcoxon test (e-h) (n = 100 (d), n = 4 (e-h); p < 0.05).
Figure 3 Figure 3
(a) Distribution des altPEP détectés par MS en fonction du nombre d'espèces de Brassicaceae dans lesquelles des homologues (plus de 80) ont été trouvés, (b) Distribution des altPEPs détectés par MS en fonction de leur cadre d'encodage, (c) Alignement des homologues d'AtDCL1alt18 (SEQ ID NO : 224) dans différentes espèces de Brassicaceae. (d, e) Conservation du premier altPEP de AP2 (d) et EIN2 (e) dans différentes espèces de Brassicaceae. (f) Activité de refAUG de EIN2 comparée à l'activité de altAUGI réalisée avec le système de transcription/translation in vitro. (a) Distribution of altPEPs detected by MS as a function of the number of Brassicaceae species in which homologs (more than 80) were found, (b) Distribution of altPEPs detected by MS as a function of their encoding frame, ( c) Alignment of AtDCL1alt18 homologs (SEQ ID NO: 224) in different Brassicaceae species. (d, e) Conservation of the first altPEP of AP2 (d) and EIN2 (e) in different species of Brassicaceae. (f) EIN2 refAUG activity compared to altAUGI activity performed with the in vitro transcription/translation system.
At : Arabidopsis thaliana, Al : Arabidopsis lyrata, Bn : Brassica napus, Bo : Brassica oleracea, Br : Brassica rapa, Bs : Boechera stricta, Cg : Capsella grandiflora, Cr : Capsella rubella, Cs : Camelina sativa, Es : Eutrema salsugineum, Rs : Raphanus sativus, Th : Thellungiella halo phi la. At: Arabidopsis thaliana, Al: Arabidopsis lyrata, Bn: Brassica napus, Bo: Brassica oleracea, Br: Brassica rapa, Bs: Boechera stricta, Cg: Capsella grandiflora, Cr: Capsella rubella, Cs: Camelina sativa, Es: Eutrema salsugineum, Rs: Raphanus sativus, Th: Thellungiella halo phi la.
Figure 4 Figure 4
Quantification par qRT PCR de l'expression de différents gènes en réponse à 100 M de différents peptides Quantification by qRT PCR of the expression of different genes in response to 100 M of different peptides
(a) Traitement des plantes avec le peptide AtCOI1alt1 ; (b) Traitement des plantes avec le peptide AtAP2alt1 ; (c) Traitement des plantes avec le peptide AtEI N2alt1 (d) Traitement des plantes avec le peptide AtCPK3alt1 . (a) Treatment of plants with the AtCOI1alt1 peptide; (b) Treatment of plants with the AtAP2alt1 peptide; (c) Treatment of plants with AtEI N2alt1 peptide (d) Treatment of plants with AtCPK3alt1 peptide.
Les barres d'erreur représentent les SEM. Error bars represent SEM.
Figure 5 Figure 5
Les peptides complémentaires augmentent l'expression des protéines Complementary peptides increase protein expression
Analyse de l'expression de la luciférase dans A. thaliana exprimant de manière constitutive le transgène LUC. (a) Gel d'agarose après amplification PCR de l'ARN obtenu par immunoprécipitation à l'aide de billes magnétiques anti-HA, suivie d'une RT-PCR, sur des plantes traitées avec le peptide indiqué (SC-HA : Scrambled cPEPluc-HA ; Luc-HA : cPEPluc- HA). (b) Expression relative du transgène de la luciférase dans les plantes traitées avec le peptide indiqué, quantifiée par RT-qPCR. (c-e) Quantification relative de LUC dans les plantes traitées avec le peptide indiqué, (f, g) Ratio entre induction et quantification de LUC dans les plantes traitées avec cPEPluc par rapport aux plantes traitées avec un Scrambled cPEPluc, en utilisant diverses concentrations de peptides (f) ou prélevé à différents points de temps (g), (h) Analyse transcriptomique des plantes traitées avec cPEPluc-HA par rapport aux plantes traitées avec Scrambled cPEPluc-HA. (i) Analyse protéomique des plantes traitées avec cPEPluc-HA par rapport aux plantes traitées avec Scrambled cPEPluc-HA. L'expression protéique a été analysée par Western blots et quantifiée avec ImageJ. Analysis of luciferase expression in A. thaliana constitutively expressing the LUC transgene. (a) Agarose gel after PCR amplification of the RNA obtained by immunoprecipitation using anti-HA magnetic beads, followed by RT-PCR, on plants treated with the indicated peptide (SC-HA: Scrambled cPEPluc-HA; Luc-HA: cPEPluc-HA). (b) Relative expression of the luciferase transgene in plants treated with the indicated peptide, quantified by RT-qPCR. (c-e) Relative quantification of LUC in plants treated with the indicated peptide, (f,g) Ratio between induction and quantification of LUC in plants treated with cPEPluc compared to plants treated with a Scrambled cPEPluc, using various peptide concentrations (f) or collected at different time points (g), (h) Transcriptomic analysis of plants treated with cPEPluc-HA compared to plants treated with Scrambled cPEPluc-HA. (i) Proteomic analysis of plants treated with cPEPluc-HA compared to plants treated with Scrambled cPEPluc-HA. Protein expression was analyzed by Western blots and quantified with ImageJ.
Les barres d'erreur représentent les SEM, les astérisques indiquent une différence significative entre la condition de test et le contrôle selon le test de Wilcoxon (a, h, i, n = 5 ; c, n = 6 ; b, d- g, n = 12 ; p < 0,05). Figure 6 Error bars represent SEMs, asterisks indicate significant difference between test condition and control according to Wilcoxon test (a, h, i, n = 5; c, n = 6; b, d- g , n = 12; p < 0.05). Figure 6
Les cPEP nécessitent une complémentarité de séquence avec leur cible pour leur activité cPEPs require sequence complementarity with their target for their activity
(a) Analyse FRET-FLIM de l'interaction entre cPEPnsp1-FAM et NSP1 (gauche) ou NSPIAcPEP (droite) in planta, (b) Quantification par qRT-PCR de l'expression de NSP1 dans les racines de M. truncatula traitées avec cPEPnspl ou Scrambled cPEPnspl . (c) Quantification via GUS des racines de M. truncatula traitées par peptide et portant la fusion Pro NSP1-NSP1-GUS. (d, e) Quantification de l'expression de NSP1 dans les feuilles de N. benthamiana après infiltration de différentes constructions : Type « sauvage » i.e. version de NSP1 dans laquelle la séquence cPEP a été supprimée (NSP1 AcPEP) ou version de NSP1 dans laquelle la séquence cPEP a été remplacée par une séquence artificielle (NSP1 AcPEP- cPEPartificiel), avec un vecteur vide (contrôle) ou cPEPnspl ou cPEPartificiel. (f, g) Formation de racines latérales de plantules de M. truncatula WT, nsp1 ou surexprimant NSP1 , en réponse à cPEPnspl ou Scrambled cPEPnspl . (h, i) Quantification des nodules sur des racines de M. truncatula traitées avec un peptide non pertinent ou cPEPskl. (j) Développement racinaire de plantules de M. truncatula infectées par A. euteiches et traitées avec un peptide non pertinent ou avec cPEPrhIO. (k) Expression relative de l'a-tubuline de A. euteiches dans des plantules de M. truncatula infectées par A. euteiches et traitées avec un peptide non pertinent ou avec cPEPrhIO. (a) FRET-FLIM analysis of the interaction between cPEPnsp1-FAM and NSP1 (left) or NSPIAcPEP (right) in planta, (b) qRT-PCR quantification of NSP1 expression in treated M. truncatula roots with cPEPnspl or Scrambled cPEPnspl. (c) Quantification via GUS of peptide-treated M. truncatula roots carrying the Pro NSP1-NSP1-GUS fusion. (d, e) Quantification of NSP1 expression in N. benthamiana leaves after infiltration of different constructs: “Wild” type i.e. version of NSP1 in which the cPEP sequence has been deleted (NSP1 AcPEP) or version of NSP1 in in which the cPEP sequence was replaced by an artificial sequence (NSP1 AcPEP- cPEPartificiel), with an empty vector (control) or cPEPnspl or cPEPartificiel. (f,g) Lateral root formation of seedlings of M. truncatula WT, nsp1 or overexpressing NSP1, in response to cPEPnspl or Scrambled cPEPnspl. (h,i) Quantification of nodules on M. truncatula roots treated with an irrelevant peptide or cPEPskl. (j) Root development of M. truncatula seedlings infected with A. Euteiches and treated with an irrelevant peptide or with cPEPrhIO. (k) Relative expression of A. Euteiches α-tubulin in M. truncatula seedlings infected with A. Euteiches and treated with an irrelevant peptide or with cPEPrhIO.
Les barres d'erreur représentent les SEM, les astérisques indiquent une différence significative entre la condition de test et le contrôle selon le test t de Student (f-i, j) ou le test de Wilcoxon (b-e, k) (b-e, k, n = 6 ; f-i, n = 50 ; j, n = 60 ; p < 0,05). Error bars represent SEMs, asterisks indicate significant difference between test condition and control according to Student's t test (f-i, j) or Wilcoxon test (b-e, k) (b-e, k, n = 6; f-i, n = 50; j, n = 60;
Figure 7 Figure 7
Les cPEPs peuvent moduler le développement d'A. thaliana et sa réponse aux stresscPEPs can modulate the development of A. thaliana and its response to stress
(a, b) Longueur des racines primaires de plantules d'A thaliana traitées avec un Scrambled cPEPdcll ou avec un cPEPdcll . (c) Teneur relative en chlorophylle de plants d'A. thaliana traités avec le peptide correspondant, (d) Reprise de la croissance des plantules de A. thaliana après un choc thermique de 45°C pendant 45 min et traitées avec le peptide correspondant, (e) Surface relative des lésions des plants de A. thaliana infectés par B. cinerea et traités avec le peptide indiqué, (f) Mesure du jour de floraison des plants d’A. thaliana traités avec les peptides indiqués, (g, h) Mesure du jour de floraison des plants d’A. thaliana traités avec un peptide non pertinent ou un mélange de cPEPs (ciblant EIN2, BRI1 , BAK1 et WUS). (i, j) Surface foliaire de plants de A. thaliana traités avec un peptide non pertinent ou un mélange de cPEPs (ciblant EIN2, BRI1 , BAK1 et WUS). Les barres d'erreur représentent les SEM, les astérisques indiquent une différence significative entre la condition testée et le contrôle selon le test t de Student (b, c, e, f, h, j) (b, n =50 ; c, e, f, h, j, n = 40 ; d, n = 5 ; p < 0,05). (a, b) Length of primary roots of A thaliana seedlings treated with Scrambled cPEPdcll or with cPEPdcll. (c) Relative chlorophyll content of A. thaliana treated with the corresponding peptide, (d) Resumption of growth of A. thaliana seedlings after a heat shock of 45°C for 45 min and treated with the corresponding peptide, (e) Relative surface area of lesions of A. thaliana plants. thaliana infected with B. cinerea and treated with the indicated peptide, (f) Measurement of the flowering day of A. thaliana treated with the indicated peptides, (g, h) Measurement of the flowering day of A. thaliana treated with an irrelevant peptide or a mixture of cPEPs (targeting EIN2, BRI1, BAK1 and WUS). (i, j) Leaf area of A. thaliana plants treated with an irrelevant peptide or a mixture of cPEPs (targeting EIN2, BRI1, BAK1 and WUS). Error bars represent SEM, asterisks indicate significant difference between tested condition and control according to Student's t test (b, c, e, f, h, j) (b, n =50; c, e, f, h, j, n = 40; d, n = 5;
Figure 8 Figure 8
Les cPEPs peuvent moduler le développement d'Æ thaliana et sa réponse aux stress (a) Quantification de l'expression de CPK3, à l'aide d'anticorps CPK3, dans des plantes de A. thaliana traitées par peptide, (b-c) Test d'infection de feuilles d'Æ thaliana traitées au peptide inoculées avec des spores de B. cinerea. cPEPs can modulate the development of A. thaliana and its response to stress (a) Quantification of CPK3 expression, using CPK3 antibodies, in peptide-treated A. thaliana plants, (b-c) Test infection of peptide-treated Æ thaliana leaves inoculated with B. cinerea spores.
Les barres d'erreur représentent les SEM, les astérisques indiquent une différence significative entre la condition de test et le contrôle selon le test de Wilcoxon (a) et le test t de Student (c) (a : n = 6 ; c, n = 50 ; p < 0,05). Error bars represent SEM, asterisks indicate significant difference between test condition and control according to Wilcoxon test (a) and Student's t test (c) (a: n = 6; c, n = 50; p < 0.05).
Figure 9 Figure 9
Les cPEP augmentent la traduction des protéines cPEPs increase protein translation
(a) Analyse de l'activité de la luciférase dans des plants d’Æ thaliana exprimant constitutivement le transgène LUC et traitées avec un Scrambled cPEPluc ou un cPEPluc, avec ou sans cycloheximide. (b) Analyse de l'activité de la luciférase après transcription/traduction in vitro de la luciférase exprimée avec une cPEPluc ou un Scrambled cPEPluc. (a) Analysis of luciferase activity in Æ thaliana plants constitutively expressing the LUC transgene and treated with Scrambled cPEPluc or cPEPluc, with or without cycloheximide. (b) Analysis of luciferase activity after in vitro transcription/translation of luciferase expressed with a cPEPluc or a Scrambled cPEPluc.
Les barres d'erreur représentent les SEM, les astérisques indiquent une différence significative entre la condition de test et le contrôle selon le test de Wilcoxon (a-b) (a, b, n = 6 ; p < 0,05). Error bars represent SEMs, asterisks indicate significant difference between test condition and control according to Wilcoxon test (a-b) (a, b, n = 6; p < 0.05).
Figure 10 Figure 10
Les cPEP sont des outils utiles en agronomie cPEPs are useful tools in agronomy
(a, b) Surface relative des lésions sur des feuilles de S. lycopersicum infectées par B. cinerea et traitées avec un peptide non pertinent ou cPEPjarl . (c, d) Résistance au stress thermique de plants de soja traités avec un peptide non pertinent ou cPEPhsp101. (e, f) Croissance (hauteur des plantes) des plants de soja traités avec un peptide non pertinent ou un mélange de cPEP (ciblant MRB1 , SHY2 et SGR1). (g, h) Surface des feuilles de plantes de B. vulgaris traitées avec un peptide non pertinent ou un mélange de cPEPs (ciblant EIN2, BRI1 , BAK1 et WUS). (i, j) Surface des feuilles de plants d'Æ hypochondriacus traités avec un peptide non pertinent ou un mélange de cPEPs (ciblant El N2, BRI1 , BAK1 et WUS). (a, b) Relative lesion area on S. lycopersicum leaves infected with B. cinerea and treated with an irrelevant peptide or cPEPjarl. (c, d) Heat stress resistance of soybean plants treated with an irrelevant peptide or cPEPhsp101. (e,f) Growth (plant height) of soybean plants treated with an irrelevant peptide or a mixture of cPEPs (targeting MRB1, SHY2, and SGR1). (g,h) Leaf surface of B. vulgaris plants treated with an irrelevant peptide or a mixture of cPEPs (targeting EIN2, BRI1, BAK1 and WUS). (i, j) Leaf surface of Æ hypochondriacus plants treated with an irrelevant peptide or a mixture of cPEPs (targeting El N2, BRI1, BAK1 and WUS).
Les barres d'erreur représentent les SEM, les astérisques indiquent une différence significative entre la condition de test et le contrôle selon le test t de Student (b, d, f, h, j, n = 40 ; p < 0,05). EXEMPLES Error bars represent SEM, asterisks indicate significant difference between test condition and control according to Student's t test (b, d, f, h, j, n = 40; p < 0.05) . EXAMPLES
MATERIELS & METHODES MATERIALS & METHODS
Matériel biologique et conditions de croissance Biological material and growth conditions
Les plants de Medicago truncatula Gaertn cv. Jemalong génotype A17 ont été cultivés sur milieu Long Aston comme décrit dans Delaux PM ét al. (New Phytol. 2013 Jul ;199(1):59-65). Les plants dArabidopsis thaliana Col-0 ont été cultivées en terre jusqu'à l'âge de 4 semaines dans une chambre de croissance (22/20°C, 16 h/8 h L/D, HR 80 %, ~ 75 pmol. m’2 s’1). Les graines de Barbarea vulgaris ont été stratifiées pendant 24h à 4°C avant d'être cultivées en pot dans une chambre de croissance. Les graines dAmaranthus hypochondriacus, Glycine max et Solanum lycopersicum ont été semées sur pots et cultivées en chambre de croissance. Les plantes Nicotiana benthamiana et M. truncatula ont été cultivées comme décrit dans Combier JP et al. (Genes and Development. 22, 1549-1559, 2008). Les plantules ABRE-LUC ont été fournies par MR Knight (Arabidopsis. Plant Cell. 23, 4079-95, 2011). Les plants dArabidopsis thaliana LUC ont été stérilisées et semées sur des plaques de 96 puits contenant 100 pL de milieu MS/2. The plants of Medicago truncatula Gaertn cv. Jemalong genotype A17 were cultured on Long Aston medium as described in Delaux PM et al. (New Phytol. 2013 Jul;199(1):59-65). Arabidopsis thaliana Col-0 plants were grown in soil until the age of 4 weeks in a growth chamber (22/20°C, 16 h/8 h L/D, RH 80%, ~ 75 pmol. m'2s'1 ) . Barbarea vulgaris seeds were stratified for 24 h at 4°C before being grown in a pot in a growth chamber. The seeds of Amaranthus hypochondriacus, Glycine max and Solanum lycopersicum were sown in pots and cultivated in a growth chamber. Nicotiana benthamiana and M. truncatula plants were grown as described in Combier JP et al. (Genes and Development. 22, 1549-1559, 2008). ABRE-LUC seedlings were provided by MR Knight (Arabidopsis. Plant Cell. 23, 4079-95, 2011). Arabidopsis thaliana LUC plants were sterilized and sown on 96-well plates containing 100 μL of MS/2 medium.
Peptides Peptides
Les peptides de séquences SEQ ID NOs : 162 à 223 et 386 à 418 ont été synthétisés par Smart Biosciences et dissous à une concentration comprise de 2 à 10 mM dans l’eau, aliquotés et conservés à - 80°C. The peptides with sequences SEQ ID NOs: 162 to 223 and 386 to 418 were synthesized by Smart Biosciences and dissolved at a concentration of 2 to 10 mM in water, aliquoted and stored at - 80°C.
Constructions plasmidiques Plasmid constructs
Les plasmides ont été obtenus en utilisant la stratégie de clonage Golden Gate dans pCAMBIA220 modifié. La fusion translationnelle Mt-NSP1 (Medtr8g020840):GUS a été réalisée en utilisant 3183 pb du promoteur NSP1 et 3180 pb de la section post CDS de NSP1. Les expressions dans les feuilles de N. benthamiana ont été réalisées en utilisant le promoteur 35S. Plasmids were obtained using the Golden Gate cloning strategy in modified pCAMBIA220. The Mt-NSP1 (Medtr8g020840):GUS translational fusion was performed using 3183 bp of the NSP1 promoter and 3180 bp of the post CDS section of NSP1. Expressions in N. benthamiana leaves were carried out using the 35S promoter.
Transformation des plantes Plant processing
Les transformations des feuilles de Nicotiana benthamiana ont été réalisées selon CombierThe transformations of the leaves of Nicotiana benthamiana were carried out according to Combier
JP et al. (Genes and Development. 22, 1549-1559, 2008). JP et al. (Genes and Development. 22, 1549-1559, 2008).
Analyse de l’expression génique Gene expression analysis
La quantification des ARNm a été réalisée par qRT-PCR en utilisant les couples de primers adéquat choisis parmi les séquences : SEQ ID NOs : 367 à 374. Les niveaux d’expression pour les contrôles ont été fixés à 100. Les primers utilisés pour les tests sur A. euteiches sont décrit dans Camborde et al. (New Phytol. 233:2232-2248. 2022). The quantification of mRNAs was carried out by qRT-PCR using the appropriate pairs of primers chosen from the sequences: SEQ ID NOs: 367 to 374. Expression levels for the controls were set at 100. The primers used for the tests on A. euteiches are described in Camborde et al. (New Phytol. 233:2232-2248. 2022).
Essais de pathogénicité Pathogenicity testing
Des feuilles d’A thaliana de type sauvage (wild-typé) ou mutant ont été pulvérisées quotidiennement avec 100 pM de peptide ou 100 pM de la version scramble correspondante pendant 3 jours. Six heures après le dernier traitement, cinq feuilles matures par plante ont été inoculées avec une gouttelette de 5 pL de 2,5x105 spores/mL de la souche B05.10 de Botrytis cinerea diluée dans 100 pM de peptide ou de sa version scramble correspondante. Après inoculation, les plantes ont été maintenues dans une humidité relative de 100 %. Ensuite, une gouttelette de 2 pL de 100 pM de peptide ou de 100 pM de la version scramble correspondante a été déposée quotidiennement sur les feuilles infectées par B. cinerea pendant 3 jours (jusqu’à l’apparition des symptômes). Les feuilles ont été prélevées des plantes pour déterminer les zones de lésions en utilisant le programme ImageJ. Pour la tomate, le protocole a été le même sauf que l'inoculation a été effectuée avec 5 000 spores de B. cinerea, sans peptide. Les peptides ont été ajoutés 1 h après l'inoculation (1 pL de 500 pM) et chaque jour pendant deux jours avec 5 pL de 100 pM de peptide. Pour l’infection de M. truncatula avec A. euteiches, les plantes ont été cultivées sur un milieu d’agar et traitées avec 10 pL de 100 pM de peptides, 24 h avant, et 24 h et 72h après l’infection avec 10 pL (1000 spores) de spores d’A. euteiches. Les plants sont récoltés 7 jours après l’inoculation pour l’extraction d’ARN. Wild-type or mutant A thaliana leaves were sprayed daily with 100 pM peptide or 100 pM of the corresponding scramble version for 3 days. Six hours after the last treatment, five mature leaves per plant were inoculated with a 5 µL droplet of 2.5x10 5 spores/mL of Botrytis cinerea strain B05.10 diluted in 100 µM peptide or its corresponding scramble version . After inoculation, plants were maintained in 100% relative humidity. Then, a 2 pL droplet of 100 pM peptide or 100 pM of the corresponding scramble version was deposited daily on B. cinerea-infected leaves for 3 days (until symptoms appeared). Leaves were collected from the plants to determine lesion areas using the ImageJ program. For the tomato, the protocol was the same except that the inoculation was carried out with 5,000 spores of B. cinerea, without peptide. Peptides were added 1 h after inoculation (1 pL of 500 pM) and each day for two days with 5 pL of 100 pM peptide. For infection of M. truncatula with A. Euteiches, plants were grown on agar medium and treated with 10 µL of 100 µM peptides, 24 h before, and 24 h and 72 h after infection with 10 pL (1000 spores) of A. Euteiches. Plants were harvested 7 days after inoculation for RNA extraction.
Traitements avec les cPEP Treatments with cPEPs
Les plants de N. benthamiana ont été traitées par pulvérisation des feuilles 24 h avant la récolte. Pour les tests Luc, 100 pL de milieu liquide MS/2 contenant les peptides ont été ajoutés à chaque puits. 5 pL de luciférine ont été ajoutés et l'activité luciférase a été lue 30 minutes plus tard à l'aide d'un spectrophotomètre. Tous les autres essais ont été réalisés en pulvérisant ou en arrosant les plantes. Pour quantifier le niveau de protéine induit par les cPEPs dans Arabidopsis, les rosettes ont été pulvérisées chaque jour avec 100 pM de peptide ou son contrôle pendant 5 jours. Les feuilles ont été récoltées 6 heures après le dernier traitement pour des analyses par western blot. En ce qui concerne le test de floraison et le contenu en chlorophylle, des plantes dArabidopsis âgées de 10 jours ont été pulvérisées avec 500 pL de 10 pM de peptide, trois fois par semaine jusqu'à la floraison. La teneur en chlorophylle a été mesurée avec le chlorophylomètre SPAD (Konica Minolta). La surface foliaire a été mesurée à l'aide du logiciel ImageJ. Les plantules de Barbarea vulgaris et A. hypochondriacus ont été traitées juste après le semis et 3 fois par semaine avec 500 pL d'un mélange de 20 pM de chaque peptide. Pour le western blot, un choc thermique a été réalisé en plaçant des plants de 20 jours cultivés dans des plaques à 24 puits sur du MS/2, 90 min à 37 °C avant de les récolter. Les plants ont été traités avec 100 pM de peptide pendant 24 h. Pour le test de résistance au choc thermique sur Æ thaliana, des plantules de 3 jours ont été traitées pendant trois jours avec 100 pM de peptide avant de placer les plantes à 45 °C pendant 45 min. Les plants ont été placés dans une chambre de croissance pour récupérer et traités 24 h après le choc thermique avec 100 pM de peptide. Pour le choc thermique du soja, des plantules d'une semaine ont été traitées 48 h, 24 h et 30 min avant d'être placées à 45 °C pendant 24 h. Pour la croissance du soja, les jeunes plants ont été traités 3 fois par semaine pendant deux semaines avec 500 pL de 100 pM de peptides. N. benthamiana plants were treated by leaf spray 24 h before harvest. For Luc assays, 100 μL of MS/2 liquid medium containing the peptides was added to each well. 5 μL of luciferin was added and luciferase activity was read 30 minutes later using a spectrophotometer. All other tests were carried out by spraying or watering the plants. To quantify the protein level induced by cPEPs in Arabidopsis, rosettes were sprayed daily with 100 pM peptide or its control for 5 days. Leaves were harvested 6 hours after the last treatment for Western blot analyses. Regarding the flowering test and chlorophyll content, 10-day-old Arabidopsis plants were sprayed with 500 µL of 10 µM peptide, three times a week until flowering. Chlorophyll content was measured with the SPAD chlorophylometer (Konica Minolta). Leaf area was measured using ImageJ software. Barbarea vulgaris and A. hypochondriacus seedlings were treated just after sowing and 3 times per week with 500 μL of a mixture of 20 μM of each peptide. For the western blot, a thermal shock was carried out by placing plants 20-day-old cultured in 24-well plates on MS/2, 90 min at 37°C before harvesting. Plants were treated with 100 pM peptide for 24 h. For the heat shock resistance test on Æ thaliana, 3-day-old seedlings were treated for three days with 100 pM peptide before placing the plants at 45°C for 45 min. Plants were placed in a growth chamber to recover and treated 24 h after heat shock with 100 pM peptide. For soybean heat shock, 1-week-old seedlings were treated for 48 h, 24 h, and 30 min before being placed at 45°C for 24 h. For soybean growth, seedlings were treated 3 times per week for two weeks with 500 µL of 100 µM peptides.
Extraction des protéines et analyses Western blot Protein extraction and Western blot analyzes
Les protéines totales des plants d’Arabidopsis ont été extraites selon Ormancey et al. (Plant Sci. 2019 Mar ;280: 12-17). Les analyses Western blot ont été réalisées selon Combier et al. (Genes Dev. 2008 Jun 1 ;22(11): 1549-59). Les niveaux de protéines ont été normalisés par rapport à la coloration Ponceau. Les anticorps ont été acquis auprès d’Agrisera et Sigma. Total proteins from Arabidopsis plants were extracted according to Ormancey et al. (Plant Sci. 2019 Mar;280:12-17). Western blot analyzes were performed according to Combier et al. (Genes Dev. 2008 Jun 1;22(11):1549-59). Protein levels were normalized to Ponceau staining. Antibodies were purchased from Agrisera and Sigma.
Transcription/traduction in vitro In vitro transcription/translation
Le système d’expression de protéines de germe de blé à haut rendement T nT® SP6 (Promega) a été utilisé selon les instructions du fabricant. La séquence LUC a été amplifiée à l’aide de la polymérase GoTaq® (Promega), les cPEP ou leurs versions Scramble correspondantes ont été ajoutés juste avant le début de la réaction. La réaction a été arrêtée après 60 min, après quoi l’activité LUC a été mesurée avec un spectrophotomètre à plaque (Perkin-Elmer Victor Nivo). The T nT® SP6 High Yield Wheat Germ Protein Expression System (Promega) was used according to the manufacturer's instructions. The LUC sequence was amplified using GoTaq® polymerase (Promega), cPEPs or their corresponding Scramble versions were added just before the start of the reaction. The reaction was stopped after 60 min, after which LUC activity was measured with a plate spectrophotometer (Perkin-Elmer Victor Nivo).
Traitement au
Figure imgf000118_0001
Treatment with
Figure imgf000118_0001
Les plants de Arabidopsis thaliana comprenant la construction LUC ont été traitées en infiltrant les feuilles avec une solution de peptide avec ou sans cycloheximide (200 pg.rnL'1) dans un milieu MS/2, 24 h avant la récolte pour le test LUC.
Figure imgf000118_0002
Arabidopsis thaliana plants comprising the LUC construct were treated by infiltrating the leaves with a solution of peptide with or without cycloheximide (200 pg.rnL' 1 ) in MS/2 medium, 24 h before harvesting for the LUC test.
Figure imgf000118_0002
La co-immunoprécipitation de l'ARN a été adaptée de Merret et al. (Plant Physiol. 174:1216- 1225. 2017). 400 mg de poudre de tissu ont été incubés dans 3 mL de tampon de lyse (200 mM Tris, pH 9,0, 110 mM acétate de potassium, 0,5% Triton X-100, 0,1 % Tween® 20, 5 mM DTT, 1 ,5% inhibiteur de protéase et 80 unités. ml-1 RNasin). Le lysat a été incubé sur la glace pendant 10 minutes, puis centrifugé à 16 000g pendant 10 minutes à 4°C. 1 ,5 mL d'extrait brut a été incubé avec 25 pL de billes magnétiques anti-HA (Thermo Scientific) pendant 1 ,5 h à 4°C sous rotation. Après liaison, les billes ont été lavées cinq fois avec 0,75 mL de tampon de lyse. L'élution a été réalisée avec 200 pL de guanidium 8 M pendant 5 minutes sur glace et précipitée pendant la nuit avec 300 pL d'éthanol à 100 %. Après centrifugation (16 000g, 45 min, 4°C), les culots ont été remis en suspension dans 200 pL de réactif Monarch de protection ADN/ARN (New England Biolabs) et l'ARN a été extrait selon les instructions du fabricant et concentré dans 10 pL à l'aide du kit de nettoyage ARN Monarch® (New England Biolabs). 300 pL de fractions d'entrée et de fractions non liées ont été conservés et l'ARN a été extrait comme décrit ci-dessus. La transcription inverse a été réalisée sur 10 pL d'éluat ou 500 ng d'entrée/sans liaison en utilisant le kit Superscript® IV (Thermo Scientific). L'amplification par PCR a été réalisée sur 1 pL d'ADNc avec des amorces spécifiques. RNA co-immunoprecipitation was adapted from Merret et al. (Plant Physiol. 174:1216-1225. 2017). 400 mg of tissue powder was incubated in 3 mL of lysis buffer (200 mM Tris, pH 9.0, 110 mM potassium acetate, 0.5% Triton X-100, 0.1% Tween® 20.5 mM DTT, 1.5% protease inhibitor and 80 units ml-1 RNasin). The lysate was incubated on ice for 10 minutes, then centrifuged at 16,000 g for 10 minutes at 4°C. 1.5 mL of crude extract was incubated with 25 μL of anti-HA magnetic beads (Thermo Scientific) for 1.5 h at 4°C under rotation. After binding, the beads were washed five times with 0.75 mL of lysis buffer. The elution was carried out with 200 μL of 8 M guanidium for 5 minutes on ice and precipitated overnight with 300 µL of 100% ethanol. After centrifugation (16,000 g, 45 min, 4°C), the pellets were resuspended in 200 μL of Monarch DNA/RNA protection reagent (New England Biolabs) and the RNA was extracted according to the manufacturer's instructions and concentrated in 10 pL using the Monarch® RNA Cleanup Kit (New England Biolabs). 300 µL of input and unbound fractions were retained and RNA was extracted as described above. Reverse transcription was performed on 10 pL of eluate or 500 ng of input/unbinding using the Superscript® IV kit (Thermo Scientific). PCR amplification was carried out on 1 pL of cDNA with specific primers.
Analyse FRET FLIM, préparation d’échantillon de feuilles pour les tests FRET FLIM analysis, leaf sample preparation for testing
Les échantillons ont été préparé sur la base des protocoles de Camborde et al (Nat Protoc. 12:1933-1950. 2017). Brièvement, la souche Agrobacterium tumefaciens GV3101 pmp90 portant les plasmides 35S-NSP1 ou 35S-NSP1AORF ont été utilisés pour infiltrer les feuilles de N. benthamiana. Les disques agro-infiltrés d'au moins trois feuilles différentes ont été fixés après 48 heures par infiltration sous vide d'une solution de fixation de paraformaldéhyde à 4 % (p/v), suivie d'une étape de perméabilisation à l'aide d'un traitement à la protéinase K. Après des lavages, la coloration des acides nucléiques a été réalisée par infiltration sous vide d'une solution de Sytox Orange 5pM (Invitrogen). Ensuite, les disques ont été lavés et montés sur TBS avant les mesures FLIM des noyaux. The samples were prepared based on the protocols of Camborde et al (Nat Protoc. 12:1933-1950. 2017). Briefly, Agrobacterium tumefaciens strain GV3101 pmp90 carrying 35S-NSP1 or 35S-NSP1AORF plasmids were used to infiltrate N. benthamiana leaves. The agro-infiltrated discs of at least three different sheets were fixed after 48 hours by vacuum infiltration of a 4% (w/v) paraformaldehyde fixing solution, followed by a permeabilization step using of treatment with proteinase K. After washing, the staining of the nucleic acids was carried out by vacuum infiltration of a 5pM Sytox Orange solution (Invitrogen). Then, the disks were washed and mounted on TBS before FLIM measurements of the nuclei.
Analyse FRET FLIM, préparation des échantillons de feuilles de cPEP pour les expériences FRET-FLIM FRET FLIM analysis, cPEP sheet sample preparation for FRET-FLIM experiments
Les plasmides 35s-NSP1 ou 35s-NSP1AcPEP ont été transformés dans la souche pmp90 de A. tumefaciens GV3101 et agro-infiltrés dans des feuilles de N. benthamiana. 40h plus tard, une solution de 10pM de cPEP-FAM a été infiltrée dans les mêmes feuilles et les plantes ont été incubées pendant 3h. Ensuite, les disques foliaires ont été fixés et traités comme décrit dans Camborde et al (Nat Protoc. 12:1933-1950. 2017), puis lavés et montés sur TBS avant les mesures de FLIM cytoplasmique. The 35s-NSP1 or 35s-NSP1AcPEP plasmids were transformed into A. tumefaciens GV3101 strain pmp90 and agro-infiltrated into N. benthamiana leaves. 40h later, a solution of 10pM cPEP-FAM was infiltrated into the same leaves and the plants were incubated for 3h. Next, leaf discs were fixed and processed as described in Camborde et al (Nat Protoc. 12:1933-1950. 2017), then washed and mounted on TBS before cytoplasmic FLIM measurements.
Analyse FRET FLIM, acquisition de données TCSPC-FLIM FRET FLIM analysis, TCSPC-FLIM data acquisition
La FLIM a été réalisée sur Leica TCS SP8 SMD qui consiste en un microscope inversé LEICA DMi8 équipé d'un système TCSPC de PicoQuant. L'excitation du donneur FITC à 470 nm a été réalisée par un laser à diode pulsé picoseconde à une fréquence de répétition de 40 MHz, à travers un objectif à immersion d'huile (63*, N. A. 1 ,4). La lumière émise a été détectée par un détecteur Leica HyD dans la gamme d'émission de 500-550 nm. Les images ont été acquises avec des photons d'acquisition allant jusqu'à 1500 par pixel. Analyse FRET FLIM, analyse des données FLIM FLIM was carried out on Leica TCS SP8 SMD which consists of a LEICA DMi8 inverted microscope equipped with a TCSPC system from PicoQuant. Excitation of the FITC donor at 470 nm was carried out by a picosecond pulsed diode laser at a repetition rate of 40 MHz, through an oil immersion objective (63*, NA 1.4). The emitted light was detected by a Leica HyD detector in the emission range of 500–550 nm. Images were acquired with acquisition photons of up to 1500 per pixel. FLIM FRET analysis, FLIM data analysis
À partir des images d'intensité de fluorescence, les courbes de décroissance ont été calculées par pixel et ajustées (par estimation du maximum de vraisemblance de la Loi de Poisson) avec un modèle de décroissance mono- ou double-exponentiel à l'aide du logiciel SymphoTime 64 (PicoQuant, Allemagne). La fonction du modèle mono-exponentiel a été appliquée pour les échantillons de donneurs avec seulement du FITC présent. La fonction modèle double- exponentielle a été utilisée pour les échantillons contenant du FITC et du Sytox. Les expériences ont été répétées au moins trois fois pour obtenir des données statistiquement valides. L'efficacité du transfert d'énergie (E) basée sur la durée de vie de la fluorescence (T) a été calculée comme suit : E = 1 - (TD+A/TD-A), OÙ TD+A est la durée de vie de la fluorescence du donneur en présence de l'accepteur et TD-A est la durée de vie de la fluorescence du donneur en l'absence de l'accepteur. From the fluorescence intensity images, the decay curves were calculated per pixel and fitted (by maximum likelihood estimation of the Poisson Law) with a mono- or double-exponential decay model using the SymphoTime 64 software (PicoQuant, Germany). The mono-exponential model function was applied for donor samples with only FITC present. The double-exponential model function was used for samples containing FITC and Sytox. The experiments were repeated at least three times to obtain statistically valid data. The energy transfer efficiency (E) based on the fluorescence lifetime (T) was calculated as follows: E = 1 - (TD+A/TD-A), WHERE TD+A is the duration lifetime of donor fluorescence in the presence of the acceptor and TD-A is the lifetime of donor fluorescence in the absence of the acceptor.
Analyses statistiques Statistical analyzes
Les valeurs moyennes de l’expression génique relative, du niveau de protéines ou des paramètres phénotypiques ont été comparées à l’aide du test de Wilcoxon ou du test t de Student. Les barres d’erreur représentent l’erreur standard de la moyenne (SEM). Les astérisques indiquent des différences significatives (p < 0,05). Mean values of relative gene expression, protein level or phenotypic parameters were compared using the Wilcoxon test or Student's t test. Error bars represent standard error of the mean (SEM). Asterisks indicate significant differences (p < 0.05).
RESULTATS RESULTS
Les altPEP contrôlent la traduction de leur gène codant altPEPs control the translation of their coding gene
Un premier objectif a été d'examiner si des altPEPs naturels pouvaient être détectés dans A. thaliana (Fig. 2a). Pour ce faire, il a été générée bio-informatiquement une liste de tous les altORFs potentiels de plus de 10 acides aminés de longueur, et situés hors cadre dans les séquences codantes de A. thaliana. Sur la base de cette liste, nous avons analysé un ensemble de données MS récemment publié (Müller J. B. et al. Nature. 582, 592-596 (2020)) et identifié 112 nouveaux altPEPs, montrant que les altPEPs sont naturellement représentés dans le protéome in planta (SEQ ID NOs : 252-363). En même temps, ont été identifiés 85 des 112 protéines de référence correspondantes dans le même ensemble de données, ce qui suggère que les altPEPs pourraient dans certains cas être plus exprimées que leurs protéines de référence. Parmi les 112 altPEP identifiées, 24 d'entre eux correspondaient au premier altORF trouvé dans la séquence codante (Fig. 2b ; SEQ ID NOs : 258, 261 , 262, 263, 270, 274, 284, 285, 286, 290, 295, 303, 312, 316, 320, 321 , 323, 324, 332, 343, 344, 352, 353 et 358). De manière surprenante, la majorité des altPEPs identifiées étaient codées à partir d'autres AUG alternatifs (altAUG), du deuxième au 23ème, ce qui montre que plusieurs altAUG, situés en aval des AUG canoniques, peuvent être traduits et être actifs in planta (Fig. 2b). La longueur moyenne des peptides codés par les altORFs détectés était de 45 acides aminés, tandis que le plus long contenait 212 acides aminés (Fig. 2c, SEQ ID NO : 295). Il est intéressant de noter que 35 altPEPs ne partageaient aucune homologie parmi les Brassicaceae (Fig. 3a). Enfin, il est surprenant que 105 altPEPs sur 112 soient codées par des altAUGs situés dans le cadre 2 (si l'on considère que la protéine principale est codée par le cadre 1) (Fig. 3b). Ces altPEPs n'ont pas été décrits par d'autres (Wang S. et al. Mol Plant 13, 1-16 (2020)), apportant des peptides traduits supplémentaires à ceux déjà décrits. Cette analyse a montré que les altPEPs sont naturellement exprimés à des niveaux détectables in planta, et qu’ils sont probablement répandus parmi les différents gènes codants annotés, bien que les altPEPs identifiés ici ne correspondent probablement qu'à une fraction des altPEPs réellement présents dans les cellules végétales. A first objective was to examine whether natural altPEPs could be detected in A. thaliana (Fig. 2a). To do this, a list of all potential altORFs of more than 10 amino acids in length, and located out of frame in the coding sequences of A. thaliana, was bioinformatically generated. Based on this list, we analyzed a recently published MS dataset (Müller JB et al. Nature. 582, 592-596 (2020)) and identified 112 novel altPEPs, showing that altPEPs are naturally represented in the proteome. in planta (SEQ ID NOs: 252-363). At the same time, 85 of the 112 corresponding reference proteins were identified in the same dataset, suggesting that altPEPs could in some cases be more expressed than their reference proteins. Among the 112 altPEPs identified, 24 of them corresponded to the first altORF found in the coding sequence (Fig. 2b; SEQ ID NOs: 258, 261, 262, 263, 270, 274, 284, 285, 286, 290, 295 , 303, 312, 316, 320, 321, 323, 324, 332, 343, 344, 352, 353 and 358). Surprisingly, the majority of altPEPs identified were encoded from other alternative AUGs (altAUG), from the second to the 23rd , which shows that several altAUGs, located downstream of canonical AUGs, can be translated and be active in planta (Fig. 2b). The average length of the peptides encoded by the detected altORFs was 45 amino acids, while the longest contained 212 amino acids (Fig. 2c, SEQ ID NO: 295). Interestingly, 35 altPEPs shared no homology among Brassicaceae (Fig. 3a). Finally, it is surprising that 105 altPEPs out of 112 are encoded by altAUGs located in frame 2 (considering that the main protein is encoded by frame 1) (Fig. 3b). These altPEPs have not been described by others (Wang S. et al. Mol Plant 13, 1-16 (2020)), providing additional translated peptides to those already described. This analysis showed that altPEPs are naturally expressed at detectable levels in planta, and are likely widespread among the different annotated coding genes, although the altPEPs identified here likely correspond to only a fraction of the altPEPs actually present in plant cells.
Pour étudier les rôles biologiques des altPEPs, deux types d'altPEPs ont été étudié : un bien conservé parmi les Brassicaceae, et correspondant à l'altORFI 8 du gène DCL1 (Fig. 3c), et un autre, correspondant au premier altORF du gène COI1, mais non conservé parmi les Brassicaceae. Des plantules d'Æ thaliana ont été traités avec le peptide AtDCLI ait 18 (SEQ I D NO : 224) et le développement des racines analysé, puisque les mutants dcl1 présentent des racines principales plus longues (Park, W., et al. Curr. Biol. 12, 1484-1495 (2002)). De manière intéressante, le traitement avec le peptide AtDCLI alt18 a conduit à une diminution du développement des racines (Fig. 2d). Une analyse par qRT-PCR et Western blot pour quantifier l'expression de DCL1 a été réalisée et alors qu'aucun effet sur l'expression de l'ARNm n'a été observé, l'application du peptide AtDCLI alt18 a augmenté l'accumulation de la protéine DCL1 (Fig. 2e, f). De manière intéressante, le traitement des plantes avec le peptide AtCOI1alt1 a révélé une augmentation du niveau de la protéine COI1 , montrant que cette propriété n'est pas limitée à DCL1 (Fig. 2g ; Fig. 4a). To study the biological roles of altPEPs, two types of altPEPs were studied: one well conserved among the Brassicaceae, and corresponding to altORFI 8 of the DCL1 gene (Fig. 3c), and another, corresponding to the first altORF of the gene COI1, but not conserved among Brassicaceae. Ae thaliana seedlings were treated with the peptide AtDCLI ait 18 (SEQ I D NO: 224) and root development analyzed, since dcl1 mutants have longer main roots (Park, W., et al. Curr. Biol. 12, 1484-1495 (2002)). Interestingly, treatment with the AtDCLI alt18 peptide led to a decrease in root development (Fig. 2d). qRT-PCR and Western blot analysis to quantify DCL1 expression was performed and while no effect on mRNA expression was observed, application of the AtDCLI alt18 peptide increased the accumulation of DCL1 protein (Fig. 2e, f). Interestingly, treatment of plants with the AtCOI1alt1 peptide revealed an increase in the level of COI1 protein, showing that this property is not limited to DCL1 (Fig. 2g; Fig. 4a).
Pour prolonger cette observation, ont été identifiés par bioinformatique les premiers altPEPs de plus de 10 acides aminés de plusieurs gènes codants bien connus de A. thaliana. Il s’agit des trois gènes : AP2 et EIN2, pour lesquels le premier altPEP potentiel était conservé parmi les Brassicaceae (Fig. 3d, e), et CPK3, pour lequel le premier altPEP n'était pas conservé. A été également testé si l'altAUG 1 du gène EIN2 est traduit in vitro, en utilisant un système de transcri ption/translation in vitro dans des extraits de germe de blé. Pour cela, le CDS de la luciférase a été fusionné au 5'UTR de EIN2 jusqu'au refAUG ou à l'altAUGI , et a été comparée l'expression de la luciférase. De manière intéressante, il a été montré que altAUGI est efficace pour promouvoir la traduction (Fig. 3f). L'efficacité de traduction est du même ordre que l'activité de refAUG, ce qui suggère une co-translation en quantité similaire de la protéine EIN2 canonique et de l'altPEP. Les plantes ont ensuite été traitées avec les trois altPEP synthétiques (AtAP2alt1 ; SEQ ID NO : 243, AtCPK3alt1 et AtEIN2alt1 ; SEQ ID NO : 241). Dans chaque cas, il a été observé une augmentation de la protéine de référence, sans détecter aucun changement dans l'expression de l'ARNm (Fig. 2g, h ; Fig. 4b, c, d). Ceci suggère que l'augmentation de l'expression des protéines de référence est probablement une propriété commune des altPEPs. En parallèle, la conservation des altPEPs ne semble pas être importante pour leur activité. Sur la base de ces deux constatations, ont été synthétisés trois altPEPs (SEQ ID NOs : 334 à 366) correspondant à différentes régions du gène EIN2 et ont été traitées indépendamment des plantes A. thaliana avec ceux-ci. De manière intéressante, les trois peptides ont été capables d'augmenter le niveau de la protéine EIN2 (Fig. 2h), montrant que tous les altPEPs testés partagent la propriété d'augmenter les niveaux de leur protéine de référence. To extend this observation, the first altPEPs of more than 10 amino acids of several well-known coding genes of A. thaliana were identified by bioinformatics. These are the three genes: AP2 and EIN2, for which the first potential altPEP was conserved among Brassicaceae (Fig. 3d, e), and CPK3, for which the first altPEP was not conserved. It was also tested whether altAUG 1 of the EIN2 gene is translated in vitro, using an in vitro transcription/translation system in wheat germ extracts. For this, the CDS of luciferase was fused to the 5'UTR of EIN2 up to refAUG or altAUGI, and the expression of luciferase was compared. Interestingly, altAUGI was shown to be effective in promoting translation (Fig. 3f). The translation efficiency is of the same order as the activity of refAUG, suggesting co-translation in similar amounts of the canonical EIN2 protein and altPEP. The plants were then treated with the three altPEPs synthetic (AtAP2alt1; SEQ ID NO: 243, AtCPK3alt1 and AtEIN2alt1; SEQ ID NO: 241). In each case, an increase in the reference protein was observed, without detecting any change in mRNA expression (Fig. 2g,h; Fig. 4b,c,d). This suggests that increased expression of reference proteins is likely a common property of altPEPs. At the same time, the conservation of altPEPs does not seem to be important for their activity. On the basis of these two findings, three altPEPs (SEQ ID NOs: 334 to 366) corresponding to different regions of the EIN2 gene were synthesized and were treated independently of A. thaliana plants with them. Interestingly, all three peptides were able to increase the level of EIN2 protein (Fig. 2h), showing that all altPEPs tested share the property of increasing the levels of their reference protein.
Les Peptides complémentaires ou cPEP Complementary Peptides or cPEPs
Il a été démontré dans le point précédent que les peptides courts peuvent interagir physiquement avec leur ARN naissant (Lauressergues D, et al. Cell Reports. 38:110339, 2022). il a été questionné si tout peptide traduit à partir d'une séquence située dans un gène codant pouvait partager la même propriété (Fig. 5a). Pour ce faire, il a été conçu un peptide complémentaire de 10 acides aminées (SEQ ID NO : 213) correspondant à un fragment de 10 acides aminés de la protéine luciférase, exprimée de façon constitutive dans Arabidopsis thaliana (Whalley et al. Plant Cell. 23, 4079-95, 2011), et ne présentant aucune homologie avec une quelconque séquence du génome de A. thaliana. En utilisant la méthode RNA-IP suivie de la PCR sur des plantes traitées avec des peptides marqués HA (cPEPluc-HA ; SEQ ID NO : 387), il a été validé qu'un tel cPEP était spécifiquement capable d'interagir avec l'ARNm de la luciférase, alors qu'un Scrambled peptide correspondant (SEQ ID NO : 386) ne le pouvait pas (Fig. 5a). Pour déterminer si cette interaction pouvait avoir une pertinence biologique, des plants ont été traités avec le cPEPluc synthétique et l'expression de la luciférase a été analysée. Alors que l'analyse qPCR n'a montré aucun effet sur l'abondance de l'ARNm (Fig. 5b), le traitement avec le peptide a augmenté l'activité de la luciférase par rapport aux traitements avec de l'eau ou des peptides non spécifiques de la luciférase (Fig. 5c). Un essai en microplaque à 96 puits a été développé, dans lequel des graines ont été semées sur un demi-milieu solide MS et les plantules de 14 jours ont été traitées en ajoutant 100 pL d’un milieu liquide contenant les peptides. Dans un second temps, dix peptides supplémentaires de 10 acides aminés ciblant des séquences distinctes du gène de la luciférase ont été développés (SEQ ID NOs : 388 à 397), dans les trois cadres (ORF1 correspond à la protéine luciférase canonique), et tous ont été capables d'augmenter l'activité luciférase (Fig. 5d). En parallèle, 7 cPEPs de 5 à 60 acides aminés (SEQ ID NOs :398 à 403) ont été conçus et synthétisés. Le traitement exogène avec ces différents peptides a révélé une augmentation de l'activité luciférase pour les peptides allant de 5 à 40 acides aminés, alors que les peptides plus longs n'ont eu aucun effet (Fig. 5e). L'effet de la concentration en peptides et du temps de traitement sur l'activité cPEPluc a ensuite été analysé. Ainsi, la concentration optimale de cPEP dans ces conditions a été trouvée à 50 pM (Fig. 5f), et 24h de traitement ont montré l'effet maximal du cPEP (Fig. 5g). Enfin, pour savoir si les cPEP pouvaient avoir des effets secondaires, des plants d’A thaliana exprimant la luciférase ont été traités avec un cPEPluc (SEQ ID NO : 213) ou son Scrambled peptide (SEQ ID NO : 220) et une analyse transcriptomique et protéomique a été effectuée (Fig. 5h, i). De manière intéressante, aucun changement dans le transcriptome et dans le protéome des plantes traitées avec le cPEPluc n’a été détecté, par rapport aux plantes traitées avec le Scrambled peptide, ce qui suggère une forte spécificité des cPEPs pour leur protéine cible. It was demonstrated in the previous point that short peptides can physically interact with their nascent RNA (Lauressergues D, et al. Cell Reports. 38:110339, 2022). it was questioned whether any peptide translated from a sequence located in a coding gene could share the same property (Fig. 5a). To do this, a complementary peptide of 10 amino acids was designed (SEQ ID NO: 213) corresponding to a fragment of 10 amino acids of the luciferase protein, expressed constitutively in Arabidopsis thaliana (Whalley et al. Plant Cell. 23, 4079-95, 2011), and showing no homology with any sequence of the genome of A. thaliana. Using the RNA-IP method followed by PCR on plants treated with HA-tagged peptides (cPEPluc-HA; SEQ ID NO: 387), it was validated that such a cPEP was specifically capable of interacting with luciferase mRNA, whereas a corresponding Scrambled peptide (SEQ ID NO: 386) could not (Fig. 5a). To determine whether this interaction could have biological relevance, plants were treated with the synthetic cPEPluc and luciferase expression was analyzed. While qPCR analysis showed no effect on mRNA abundance (Fig. 5b), treatment with the peptide increased luciferase activity compared to treatments with water or non-specific luciferase peptides (Fig. 5c). A 96-well microplate assay was developed, in which seeds were sown on half MS solid medium and 14-day-old seedlings were treated by adding 100 µL of liquid medium containing the peptides. In a second step, ten additional peptides of 10 amino acids targeting distinct sequences of the luciferase gene were developed (SEQ ID NOs: 388 to 397), in the three frames (ORF1 corresponds to the canonical luciferase protein), and all were able to increase luciferase activity (Fig. 5d). In parallel, 7 cPEPs of 5 to 60 amino acids (SEQ ID NOs: 398 to 403) were designed and synthesized. Exogenous treatment with these different peptides revealed a increased luciferase activity for peptides ranging from 5 to 40 amino acids, whereas longer peptides had no effect (Fig. 5e). The effect of peptide concentration and treatment time on cPEPluc activity was then analyzed. Thus, the optimal concentration of cPEP under these conditions was found to be 50 pM (Fig. 5f), and 24 h of treatment showed the maximum effect of cPEP (Fig. 5g). Finally, to find out if cPEPs could have side effects, A thaliana plants expressing luciferase were treated with a cPEPluc (SEQ ID NO: 213) or its Scrambled peptide (SEQ ID NO: 220) and a transcriptomic analysis and proteomics was performed (Fig. 5h,i). Interestingly, no changes in the transcriptome and proteome of plants treated with cPEPluc were detected, compared to plants treated with Scrambled peptide, which suggests a strong specificity of cPEPs for their target protein.
Généralisation du concept de peptides augmentant l'expression des protéines Generalization of the concept of peptides increasing protein expression
Afin de vérifier si les cPEP peuvent cibler n'importe quelle protéine, il a été étudié et validé l'activité de 12 cPEP ciblant 12 protéines différentes pour lesquelles des anticorps étaient disponibles, dans trois espèces végétales différentes. La question était de savoir si l'augmentation de la quantité de protéines observée après le traitement par les cPEP était suffisante pour moduler le développement des plantes. Pour répondre à cette question, le développement s’est concentré sur différentes protéines de la plante modèle M. truncatula. L'application de cPEPnspl (SEQ ID NO :164) a diminué la quantité de racines latérales (Fig. 6f, g), tout comme le mutant nsp1 et une surexpression de NSP1 , alors que tous deux sont restés insensibles au peptide (Fig. 6g). Le gène Sickle (SKL) de M. truncatula est homologue à AtEIN2, et les mutants ski développent plus de nodules que les plantes de type sauvage (Penmetsa étal. Plant J. 55:580-595, 2008). De manière cohérente, l'application d'un cPEPskl (SEQ ID NO : 411) a conduit à une diminution de la quantité de nodules (Fig. 6h, i). Il a ensuite été ciblé la protéine RH 10 de M. truncatula, qui module la réponse des plantes à un oomycète pathogène, Aphanomyces euteiches (Camborde ét al. New Phytol. 233:2232-2248. 2022). Le traitement des plantes avec un cPEP ciblant MtRH10 (SEQ ID NO : 412) a augmenté la résistance des plantes au pathogène, comme le révèle l'augmentation du développement des racines et la diminution de l'expression de l'a-tubuline d'A euteiches dans les racines (Fig. 6j, k). To test whether cPEPs can target any protein, the activity of 12 cPEPs targeting 12 different proteins for which antibodies were available was studied and validated, in three different plant species. The question was whether the increase in protein quantity observed after cPEP treatment was sufficient to modulate plant development. To answer this question, development focused on different proteins from the model plant M. truncatula. Application of cPEPnspl (SEQ ID NO:164) decreased the amount of lateral roots (Fig. 6f,g), as did the nsp1 mutant and overexpression of NSP1, while both remained insensitive to the peptide (Fig. 6g). The Sickle (SKL) gene of M. truncatula is homologous to AtEIN2, and ski mutants develop more nodules than wild-type plants (Penmetsa et al. Plant J. 55:580-595, 2008). Consistently, the application of a cPEPskl (SEQ ID NO: 411) led to a decrease in the amount of nodules (Fig. 6h, i). The RH 10 protein of M. truncatula was then targeted, which modulates the response of plants to a pathogenic oomycete, Aphanomyces Euteiches (Camborde et al. New Phytol. 233:2232-2248. 2022). Treatment of plants with a cPEP targeting MtRH10 (SEQ ID NO: 412) increased plant resistance to the pathogen, as revealed by increased root development and decreased α-tubulin expression. A Euteiches in the roots (Fig. 6j, k).
Généralisation à d’autre modèle de plantes, d’autres protéines Generalization to other plant models, other proteins
Les travaux se sont ensuite concentrés sur différentes protéines de A. thaliana impliquées dans différentes fonctions de la plante. Tout d'abord, le traitement de plantules d'A. thaliana avec un cPEPdcll (SEQ ID NO : 163) a conduit à une diminution de la croissance des racines primaires puisque les mutants dcl1 présentent des racines primaires plus longues (Park ét al. Curr. Biol. 12:1484-1495 (2002). (Fig. 7a, b). Puis des plants d'Æ thaliana ont été traités avec des cPEP ciblant des régulateurs de la teneur en chlorophylle. De façon intéressante, un cPEP ciblant la protéine ABI5 (SEQ ID NO : 406) a pu être identifié, diminuant la teneur en chlorophylle, et un cPEP ciblant la protéine SGR1 (SEQ ID NO : 407), augmentant cette teneur (Fig. 7c). De même, un cPEP ciblant la protéine HSP101 (SEQ ID NO : 404), impliquée dans la tolérance au stress thermique (Queitsch et al, Plant Cell. 12:479-492, 2000), a amélioré la viabilité des plantules à un choc thermique (Fig. 7d). En parallèle, plusieurs régulateurs de défense des plants d'Æ thaliana ont été ciblés, AGB1 (SEQ ID NO : 195), MAPKKK3 (SEQ ID NO : 187), JAR1 (SEQ ID NO : 192), MAPKKK5 (SEQ ID NO : 188), ABCG34 (SEQ ID NO : 194) et CPK3 (SEQ ID NO : 162). De manière intéressante, ces cPEPs ont amélioré la défense des plantes contre le champignon nécrotrophe Botrytis cinerea, comme le révèle la diminution de la taille des lésions observée dans les plantes traitées avec les cPEPs par rapport aux plantes traitées avec un peptide non pertinent (Fig. 7e ; Fig. 8). Ensuite, plusieurs cPEPs ont été conçu ciblant différentes protéines impliquées dans le développement des plants en mesurant le jour de floraison. Il a pu être identifié des cPEPs augmentant le développement des plantes (SHY2 et MRB1 , SEQ ID NOs : 178 et 180) alors que d'autres étaient capables de diminuer le développement des plantes (BRI1 , SEQ ID NO 198 ; BAK1 , SEQ ID NO 199 ; TAP46, SEQ ID NO 181 ; SPT, SEQ ID NO 182 ; EIN2, SEQ ID NO 204 ; GA2OX7, SEQ ID NO 183 ; PHYB, SEQ ID NO 184 ; HAG5, SEQ ID NO 185 ; SHR, SEQ ID NO 186 et WUS, SEQ ID NO 196) (Fig. 7f). Enfin, il a été cherché à savoir si les cPEPs pouvaient avoir des effets synergiques, en mélangeant certains d'entre eux. De manière intéressante, alors que chaque peptide séparément a diminué le développement jusqu'à 17% (Fig. 7f), un mélange de cPEPs ciblant EIN2, BRI1 , BAK1 et WUS (respectivement SEQ ID NOs : 204, 198, 199 et 196), a diminué le développement de 23%, comme le montre le jour de floraison (Fig. 7g, h) et la croissance des feuilles (Fig. 7i, j), montrant un effet synergique des cPEPs. Toutes ces données ont montré que, en plus d'améliorer l'expression des protéines, les cPEPs sont des outils utiles pour moduler précisément plusieurs phénotypes végétaux. The work then focused on different A. thaliana proteins involved in different functions of the plant. First of all, the treatment of seedlings of A. thaliana with a cPEPdcll (SEQ ID NO: 163) led to a reduction in the growth of primary roots since dcl1 mutants present longer primary roots (Park et al. Curr. Biol. 12:1484-1495 (2002). (Fig. 7a, b). Then Ae thaliana plants were treated with cPEPs targeting regulators of chlorophyll content. Interestingly, a cPEP targeting the ABI5 protein (SEQ ID NO: 406) could be identified, reducing the chlorophyll content, and a cPEP targeting the SGR1 protein (SEQ ID NO: 407), increasing this content (Fig. 7c ). Likewise, a cPEP targeting the HSP101 protein (SEQ ID NO: 404), involved in tolerance to heat stress (Queitsch et al, Plant Cell. 12:479-492, 2000), improved the viability of seedlings to shock. thermal (Fig. 7d). In parallel, several defense regulators of Æ thaliana plants were targeted, AGB1 (SEQ ID NO: 195), MAPKKK3 (SEQ ID NO: 187), JAR1 (SEQ ID NO: 192), MAPKKK5 (SEQ ID NO: 188), ABCG34 (SEQ ID NO: 194) and CPK3 (SEQ ID NO: 162). Interestingly, these cPEPs improved plant defense against the necrotrophic fungus Botrytis cinerea, as revealed by the decrease in lesion size observed in plants treated with cPEPs compared to plants treated with an irrelevant peptide (Fig. 7e; Then, several cPEPs were designed targeting different proteins involved in plant development by measuring the flowering day. It was possible to identify cPEPs increasing plant development (SHY2 and MRB1, SEQ ID NOs: 178 and 180) while others were capable of reducing plant development (BRI1, SEQ ID NO 198; BAK1, SEQ ID NO 199; SEQ ID NO 181; EIN2, SEQ ID NO 204; 186 and WUS, SEQ ID NO 196) (Fig. 7f). Finally, it was sought to find out if cPEPs could have synergistic effects, by mixing some of them. Interestingly, while each peptide separately decreased development by up to 17% (Fig. 7f), a mixture of cPEPs targeting EIN2, BRI1, BAK1 and WUS (respectively SEQ ID NOs: 204, 198, 199 and 196) , decreased development by 23%, as shown in flowering day (Fig. 7g, h) and leaf growth (Fig. 7i, j), showing a synergistic effect of cPEPs. All these data showed that, in addition to improving protein expression, cPEPs are useful tools to precisely modulate several plant phenotypes.
Les cPEP augmentent l'efficacité de la traduction des protéines cPEPs increase the efficiency of protein translation
Les cPEPs augmentent la quantité de protéines sans perturber les niveaux d'ARNm, ce qui suggère que les cPEPs augmentent la traduction ou la stabilité des protéines. Afin de distinguer les deux options, des plants de A. thaliana exprimant le gène LUC ont été traités avec cPEPluc (SEQ ID NO : 213) et du cycloheximide (CHX), un inhibiteur de traduction. Le dosage de l'activité luciférase a montré que l'effet du cPEPluc était inhibé en présence de CHX, ce qui montre que les cPEP n'agissent pas sur la stabilité des protéines (Fig. 9a). Pour appuyer ces données, le gène LUC a été exprimé avec ou sans cPEPluc dans le système de transcription/traduction in vitro du germe de blé, où aucune activité protéasique ne se produit. Cela a révélé que la cPEPluc augmentait l'activité de LUC in vitro, renforçant fortement l'idée que les cPEP augmentent l'efficacité de la traduction des protéines (Fig. 9b). cPEPs increase the amount of protein without disrupting mRNA levels, suggesting that cPEPs increase protein translation or stability. To distinguish between the two options, A. thaliana plants expressing the LUC gene were treated with cPEPluc (SEQ ID NO: 213) and cycloheximide (CHX), a translation inhibitor. Luciferase activity assay showed that the effect of cPEPluc was inhibited in the presence of CHX, showing that cPEPs do not act on protein stability (Fig. 9a). To support these data, the LUC gene was expressed with or without cPEPluc in the wheat germ in vitro transcription/translation system, where no protease activity occurs. This revealed that cPEPluc increased LUC activity in vitro, strongly supporting the idea that cPEPs increase protein translation efficiency (Fig. 9b).
Intérêt agronomique des cPEP Agronomic interest of cPEPs
L'intérêt principal des cPEPs pourrait être l'utilisation en agronomie pour améliorer le rendement des cultures. Pour en apporter la preuve, ils ont été testés sur des plantes d'intérêt agronomique, en se concentrant sur les mêmes phénotypes que ceux qui ont été étudiés sur les plantes modèles. Ainsi, les travaux se sont d'abord concentrés sur la défense des plantes et la protéine JAR1 de la tomate a été ciblée. Conformément à l'observation précédente chez A. thaliana, le traitement de la tomate avec cPEPjarl (SEQ ID NO : 413) a permis d'améliorer la résistance de la plante à B. cinerea (Fig. 10a, b). En parallèle, il a été identifié l'homologue de la HSP101 dans le soja et un cPEP ciblant cette protéine a été conçu (SEQ ID NO : 408). De manière intéressante, le traitement des plantes de soja avec ce peptide a augmenté leur tolérance à un stress thermique (Fig. 10c, d). En parallèle, il a été validé sur le soja que l'utilisation de cPEP peut améliorer la croissance des plantes, en utilisant un mélange de cPEP ciblant SHY2, MRB1 et SGR1 (respectivement SEQ ID NOs : 410, 409 et 407) (Fig. 10e, f). Enfin, il a été testé si les cPEPs pouvaient diminuer la croissance des adventices (mauvaises herbes) en ciblant une espèce de Brassicaceae, Barbarea vulgaris, et il a été montré qu'un mélange de cPEPs ciblant EIN2, BRI1 , BAK1 et WUS (respectivement SEQ ID NOs : 417, 415, 414 et 416), était capable de le faire (Fig. 10g, h). Pour aller plus loin, il a été sélectionné une des mauvaises herbes les plus envahissantes et problématiques, VAmaranthus, et il a été conçu des cPEPs pour cibler les protéines correspondantes (respectivement SEQ ID NOs : 204, 176, 177 et 196). Un mélange de ces cPEPs a été capable de diminuer la croissance de la plante (Fig. 10i, j). The main interest of cPEPs could be the use in agronomy to improve crop yield. To provide proof of this, they were tested on plants of agronomic interest, focusing on the same phenotypes as those that were studied on model plants. Thus, the work first focused on plant defense and the tomato JAR1 protein was targeted. Consistent with the previous observation in A. thaliana, treatment of tomato with cPEPjarl (SEQ ID NO: 413) improved the plant's resistance to B. cinerea (Fig. 10a, b). In parallel, the homolog of HSP101 in soybean was identified and a cPEP targeting this protein was designed (SEQ ID NO: 408). Interestingly, treatment of soybean plants with this peptide increased their tolerance to heat stress (Fig. 10c, d). In parallel, it was validated on soybean that the use of cPEP can improve plant growth, using a mixture of cPEPs targeting SHY2, MRB1 and SGR1 (respectively SEQ ID NOs: 410, 409 and 407) (Fig. 10th, f). Finally, it was tested whether cPEPs could decrease the growth of weeds by targeting a Brassicaceae species, Barbarea vulgaris, and it was shown that a mixture of cPEPs targeting EIN2, BRI1, BAK1 and WUS (respectively SEQ ID NOs: 417, 415, 414 and 416), was capable of doing so (Fig. 10g, h). To go further, one of the most invasive and problematic weeds, Amaranthus, was selected and cPEPs were designed to target the corresponding proteins (respectively SEQ ID NOs: 204, 176, 177 and 196). A mixture of these cPEPs was able to decrease plant growth (Fig. 10i, j).
Conclusion Conclusion
Les résultats présentés ont démontré la possibilité de moduler l'expression de n'importe quel gène codant par application externe de petits peptides synthétiques, facilitant ainsi l'étude des gènes sans avoir besoin de plantes transgéniques. Cela peut être particulièrement pertinent dans le cas de plantes récalcitrantes à la transformation génétique. Le simple fait d'arroser ou de pulvériser les plantes avec des cPEP permet une réponse biologique conforme à ce qui est connu de la fonction des protéines ciblées, comme la modulation de la croissance des plantes ou l'amélioration de la résistance des plantes à certains pathogènes. The results presented demonstrated the possibility of modulating the expression of any coding gene by external application of small synthetic peptides, thus facilitating the study of genes without the need for transgenic plants. This may be particularly relevant in the case of plants that are recalcitrant to genetic transformation. Simply watering or spraying plants with cPEPs enables a biological response consistent with what is known about the function of targeted proteins, such as modulating plant growth or enhancing plant resistance to certain pathogens.
L'agriculture du 21ème siècle est confrontée à plusieurs défis de taille pour nourrir la population mondiale croissante. Dans ce contexte, il est urgent de trouver de nouvelles molécules pour maintenir ou améliorer le rendement des cultures. Jusqu'à présent, aucune alternative crédible aux produits chimiques n'a émergé. L'utilisation de CRISPR en agriculture est prometteuse, notamment pour améliorer la croissance des cultures, mais il est difficile de penser à la lutte contre les mauvaises herbes avec cette stratégie. L'utilisation de petits ARN, malgré son fantastique potentiel, est confrontée à l'insoluble problème de la faible pénétration dans les cellules végétales, conduisant à une faible activité dans les champs, sauf pour le contrôle des insectes. En parallèle, il a été montré ici que les cPEP sont capables de moduler différents traits des plantes, tels que la résistance à la chaleur ou la teneur en chlorophylle, qui sont très difficiles à gérer avec des produits chimiques ou d'autres molécules. 21st century agriculture faces several significant challenges in feeding the world's growing population. In this context, it is urgent to find new molecules to maintain or improve crop yield. So far, no credible alternatives to chemicals have emerged. The use of CRISPR in agriculture is promising, especially for improving crop growth, but it is difficult to think about weed control with this strategy. The use of small RNAs, despite its fantastic potential, faces the insoluble problem of low penetration into plant cells, leading to low activity in fields, except for insect control. In parallel, it was shown here that cPEPs are capable of modulating different plant traits, such as heat resistance or chlorophyll content, which are very difficult to manage with chemicals or other molecules.
Dans ce contexte, le développement de la technologie des cPEPs ouvre une nouvelle voie en agriculture avec l'utilisation de petits peptides. En parallèle, le mode d'action des cPEP, basé sur la complémentarité de leur séquence avec la protéine ciblée, permettra d'identifier plus facilement les interactions non-spécifique par bio-informatique, ce qui permettra de cibler une seule espèce, une famille ou toutes les plantes. In this context, the development of cPEPs technology opens a new path in agriculture with the use of small peptides. In parallel, the mode of action of cPEPs, based on the complementarity of their sequence with the targeted protein, will make it possible to more easily identify non-specific interactions by bioinformatics, which will make it possible to target a single species, a family. or all plants.
Enfin, les peptides étant de courts polymères d'acides aminés, ils sont susceptibles d'être rapidement dégradés par le microbiote du sol, contrairement aux produits chimiques polluants. Par ailleurs, la pénétration des peptides dans les cellules animales semble difficile sans la présence de peptides pénétrant dans les cellules ce qui laisse penser que les cPEP n'auront pas d'activité biologique chez l'animal et l'homme, hormis une éventuelle toxicité intrinsèque. Finally, as peptides are short polymers of amino acids, they are likely to be quickly degraded by the soil microbiota, unlike polluting chemicals. Furthermore, the penetration of peptides into animal cells seems difficult without the presence of peptides penetrating into cells, which suggests that cPEPs will have no biological activity in animals and humans, apart from possible toxicity. intrinsic.

Claims

REVENDICATIONS Procédé de préparation et de détermination d’un cPEP, ledit cPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés ; étant capable de moduler l’accumulation d’une protéine dans une cellule végétale ; et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine, ledit procédé comprenant : a. une étape de détermination de la séquence d’acides nucléiques de l’ARN messager (ARNm) codant ladite protéine ; b. une étape de détermination au sein de cet ARNm de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale et qui correspond au cadre ouvert de lecture codant ladite protéine ; c. une étape de détermination au sein de cette séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale d’un fragment de celle-ci, ledit fragment ayant une taille de 3n nucléotides, n étant compris de 4 à 70, en particulier n étant compris de 4 à 41 , et ledit fragment ayant une taille inférieure à celle de la séquence d’acides nucléiques naturellement traduite dans ladite cellule végétale ; d. une étape de production du peptide codé par ledit fragment ; et e. une étape de comparaison : CLAIMS Process for preparing and determining a cPEP, said cPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids; being capable of modulating the accumulation of a protein in a plant cell; and not being capable of modulating the accumulation of the mRNA encoding said protein, said method comprising: a. a step of determining the nucleic acid sequence of the messenger RNA (mRNA) encoding said protein; b. a step of determining within this mRNA the nucleic acid sequence naturally translated in said plant cell and which corresponds to the open reading frame encoding said protein; vs. a step of determining within this nucleic acid sequence naturally translated in said plant cell a fragment thereof, said fragment having a size of 3n nucleotides, n being comprised from 4 to 70, in particular n being comprised from 4 to 41, and said fragment having a size smaller than that of the nucleic acid sequence naturally translated in said plant cell; d. a step of producing the peptide encoded by said fragment; summer. a comparison step:
- entre l’accumulation de ladite protéine dans une cellule végétale en présence dudit peptide et l’accumulation de ladite protéine dans une cellule végétale de même type en absence dudit peptide ; et/ou - between the accumulation of said protein in a plant cell in the presence of said peptide and the accumulation of said protein in a plant cell of the same type in the absence of said peptide; and or
- entre le phénotype d’une plante en présence dudit peptide et le phénotype d’une plante de même type en absence dudit peptide, dans laquelle : - between the phenotype of a plant in the presence of said peptide and the phenotype of a plant of the same type in the absence of said peptide, in which:
- une différence de la quantité de ladite protéine en présence dudit peptide par rapport à la quantité de ladite protéine en absence dudit peptide ; et/ou - a difference in the quantity of said protein in the presence of said peptide compared to the quantity of said protein in the absence of said peptide; and or
- une différence du phénotype en présence dudit peptide par rapport au phénotype en absence dudit peptide, indique que ledit peptide est un cPEP capable de moduler l’accumulation de ladite protéine dans une cellule végétale. cPEP, de 4 à 70 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. Acide nucléique codant un cPEP selon la revendication 2. Composition comprenant un cPEP en tant que substance active, ledit cPEP : ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm ; étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine ; et étant notamment à une concentration comprise de 5 pM à 500 pM ou de 30 pM à 70 pM, ou étant notamment à une concentration de 50 pM. Composition selon la revendication 4, ladite composition étant une composition phytopharmaceutique, une composition herbicide ou une composition d’enrobage, en particulier ladite composition d’enrobage comprenant en outre au moins un agent de fixation. Semence enrobée comprenant une graine de plante, ladite graine de plante étant enrobée par une composition d’enrobage selon la revendication 5. Utilisation d’un cPEP en tant qu’agent phytosanitaire pour moduler l’accumulation d’une protéine dans une cellule végétale, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. Procédé de modulation de l’accumulation d’une protéine dans une cellule végétale comprenant une étape d’introduction : d’un cPEP ; ou d’un acide nucléique codant ledit cPEP et les moyens de l’exprimer, dans ladite cellule végétale, l’introduction dudit cPEP entraînant une modulation de la quantité de ladite protéine dans ladite cellule végétale, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture de ladite protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. Procédé selon la revendication 8, ledit procédé permettant : de favoriser le développement d’une plante ; ou de ralentir ou d’empêcher le développement d’une plante. Plante modifiée comprenant un cPEP introduit par voie exogène, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. Plante transgénique comprenant un acide nucléique codant un cPEP et les moyens de l’exprimer, ledit cPEP ayant une taille comprise de 4 à 70 acides aminés, en particulier de 4 à 41 acides aminés, dont la séquence d’acides aminés correspond à la traduction via le code génétique d’un fragment d’une séquence d’acides nucléiques naturellement traduite sur un ARNm dans une cellule végétale laquelle correspond au cadre ouvert de lecture d’une protéine codée par ledit ARNm, ledit cPEP étant capable de moduler l’accumulation de ladite protéine dans la cellule végétale et n’étant pas capable de moduler l’accumulation de l’ARNm codant ladite protéine. - a difference in the phenotype in the presence of said peptide compared to the phenotype in the absence of said peptide indicates that said peptide is a cPEP capable of modulating the accumulation of said protein in a plant cell. cPEP, from 4 to 70 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open frame reading a protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein. Nucleic acid encoding a cPEP according to claim 2. Composition comprising a cPEP as active substance, said cPEP: having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, including the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA; being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein; and being in particular at a concentration of 5 pM to 500 pM or from 30 pM to 70 pM, or being in particular at a concentration of 50 pM. Composition according to claim 4, said composition being a phytopharmaceutical composition, a herbicidal composition or a coating composition, in particular said coating composition further comprising at least one fixing agent. Coated seed comprising a plant seed, said plant seed being coated with a coating composition according to claim 5. Use of a cPEP as a phytosanitary agent for modulating the accumulation of a protein in a plant cell, said cPEP having a size comprised from 4 to 70 amino acids, in particular from 4 to 41 amino acids, the sequence of which of amino acids corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein. Method for modulating the accumulation of a protein in a plant cell comprising a step of introducing: a cPEP; or a nucleic acid encoding said cPEP and the means of expressing it, in said plant cell, the introduction of said cPEP leading to a modulation of the quantity of said protein in said plant cell, said cPEP having a size between 4 and 70 amino acids, in particular from 4 to 41 amino acids, whose amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of said protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein. Method according to claim 8, said method making it possible to: promote the development of a plant; or to slow or prevent the development of a plant. Modified plant comprising a cPEP introduced exogenously, said cPEP having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, of which the amino acid sequence corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated on an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein. Transgenic plant comprising a nucleic acid encoding a cPEP and the means for expressing it, said cPEP having a size of from 4 to 70 amino acids, in particular from 4 to 41 amino acids, the amino acid sequence of which corresponds to the translation via the genetic code of a fragment of a nucleic acid sequence naturally translated onto an mRNA in a plant cell which corresponds to the open reading frame of a protein encoded by said mRNA, said cPEP being capable of modulating the accumulation of said protein in the plant cell and not being capable of modulating the accumulation of the mRNA encoding said protein.
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